Electron-spin relaxation phenomena in irradiated saccharides detected by pulsed electron paramagnetic resonance spectroscopy

Radiation Physics and Chemistry 81 (2012) 1639–1645

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Electron-spin relaxation phenomena in irradiated saccharides detected by pulsed electron paramagnetic resonance spectroscopy Masahiro Kikuchi a,n, Hiromi Kameya b, Yuhei Shimoyama a, Mitsuko Ukai c, Yasuhiko Kobayashi a a

Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan c Hokkaido University of Education, 1-2 Hachiman-cho, Hakodate, Hokkaido 040-8567, Japan b

H I G H L I G H T S c c c c c

Field-swept spectra of irradiated sugars were recorded by pulsed EPR spectroscopy. Relaxation times of unpaired electrons were directly determined at each peak. The relaxation times of side peaks were longer than those of main peaks. T1 values of irradiated sugars show no correlation with the irradiation dose. The T2 values become shorter relaxation times by the increasing doses.

a r t i c l e i n f o

abstract

Article history: Received 19 August 2011 Accepted 10 May 2012 Available online 18 May 2012

We measured the relaxation times of radicals in saccharides upon g-irradiation by means of X-band pulsed electron paramagnetic resonance (EPR) spectroscopy. We found that the field-swept signal of irradiated fructose by pulsed EPR showed three to four peaks depending on the dose. The relaxation times (T1 and T2) of the side peaks were longer than those of the main peak(s) from each irradiation, indicating that the radicals showing side peaks interact less with the surrounding environment. From relaxation time measurements of several irradiated saccharides, we conclude that T2 relaxation times decrease with the increasing irradiation dose. In contrast, T1 relaxation times show no correlation with the irradiation dose. & 2012 Elsevier Ltd. All rights reserved.

Keywords: Pulsed EPR detection g-Irradiation Relaxation time Hyperfine interaction

1. Introduction During the last two decades, food irradiation has been performed by using g-rays, X-rays, and electron beams. This process is an easy, clean, and secure technology that reduces spoilage loss and improves hygienic quality. In this context, several methods have been discussed to detect the radiation processing of foodstuffs. Among them, electron spin resonance (ESR) spectroscopy is the leading method for identification of specific classes of irradiated foods. Ten protocols were adopted by the European Committee of Standardization (CEN), of which three employed the ESR method. These deal with bone-containing meat (EN1786, 1997), cellulose-containing foodstuffs (EN1787, 2000; Ukai and Shimoyama, 2003), and foodstuffs that contain saccharides (EN13708, 2001).

n

Corresponding author. Tel.: þ81 27 346 9542; fax: þ 81 27 346 9688. E-mail address: [email protected] (M. Kikuchi).

0969-806X/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2012.05.010

The research group of Callens et al. (De Cooman et al., 2008; Tarpan et al., 2008; Vanhaelewyn et al., 2006) employed an irradiated single-crystal saccharide and studied the angular dependency of its electron paramagnetic resonance (EPR) signal. They further determined and identified the EPR signal of the radical by the electron nuclear double resonance (ENDOR) technique. Using density functional theory (DFT), they determined and compared the structures for the radical and crystal. Yordanov and Karakirova (2007a, 2007b) studied sucrose powder as a food irradiation dosimeter and determined the time-dependent variation of the dose response and EPR signal intensities. They also determined and compared the signal intensities of UV absorption and EPR signals. Karakirova et al. (2010) investigated the effects of irradiation on saccharides and observed a decrease in T2 as the dose increased. In contrast, they found that T1 values did not correlate with the irradiation dose. Currently, there are two EPR methods to detect spin information in the paramagnetic system. One is the continuous wave (CW)-EPR method (Alger, 1968; Brunel, 1996), which irradiates a weak continuous microwave without disturbing the steady-state

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spin system. The other is the pulsed EPR method, which employs instantaneous microwave radiation and reorients spin systems observable in the absence of a microwave field (Calle et al., 2006). In the present study, we employed electron spin-echo (ESE) signals in terms of pulsed EPR methodology, i.e., the p/2- and p-pulse sequence. We measured various magnetic parameters such as the g-value and hyperfine couplings. Both parameters provided information on the structure as well as the strength of interaction. Based on the spin-echo method, we further determined the spin relaxation times. During the spin-echo detection of radicals in saccharide, we identified an electron spin-echo envelope modulation (ESEEM) signal that may provide information on nuclear hyperfine interactions. We analyze the hyperfine interactions in the vicinity of radical centers.

Research Institute of Japan Atomic Energy Agency. We used g-ray irradiation originated from 60Co as the radiation source. Irradiation doses of 1, 10, 25, 50, and 100 kGy were applied by changing both the irradiation time and the distance from the source. Typically, 100 kGy was given by irradiation for 16 h at 22.7 cm (a dose rate of 6.25 kGy/h). 2.3. EPR measurements

The specimens were various saccharides purchased from domestic chemical companies: i.e., fructose (Junsei Chemical Co., Ltd., Tokyo); lactose, glucose, and sucrose (Wako Pure Chemical Industries, Osaka); and maltose and galactose (Kanto Chemical Co., Inc., Tokyo). After g-irradiation, a specimen was carefully sealed in a quartz sample tube (99.9% purity; JEOL, Akishima). EPR measurements were then carried out by using pulsed EPR spectroscopy.

CW-EPR measurements were performed with an RE-3X (JEOL) at 300 K by using the microwave X-band frequency (9.4 GHz). The field modulation frequency was 100 kHz. Both an NMR Field Meter ESFC5 (JEOL, Akishima) and a microwave frequency counter TR5212 (ADVANTEST, Tokyo) were used to determine the exact measurement conditions. For pulsed EPR measurements, we first attempted to detect the pulsed signals of the electron spin-echo signals. By using a pulsed EPR spectrometer (ESP-380E, Bruker Biospin), we detected spinecho signals with a p/2–p pulse sequence. We set the p/2 pulse to 16 ns, and the p pulse to 24 ns. The p pulse was assigned to be 32 ns when the p/2 pulse was assigned to be 16 ns. However, in practice, we used the shorter pulse (i.e., 24 ns) to compensate the pulse quality of the pin diode. The pulse interval was 200 ns and the recycle delay was 1 ms. For field-swept detection by pulsed EPR, we employed simultaneous echo detection at various resonance fields. In this way, an EPR spectrum similar to the absorption signal instead of the first derivative signal may be observed.

2.2. Irradiation treatments

2.4. Observation of relaxation times via the spin-echo method

All irradiation treatments were carried out at room temperature (ca 300 K) in a facility of the Takasaki Advanced Radiation

p/2–t–p pulse sequences of 16 ns and 24 ns. The pulse interval

2. Materials and methods 2.1. Materials

S1

M1

M2

S2 100 kGy

100 kGy

M1 Signal Intensity (AU)

Based on the echo measurement method, we utilized two

25 kGy

M2

S1

25 kGy S2

M1 1 kGy

1 kGy S1

S2

337

344 Magnetic Field (mT)

3 32

34 1 Magnetic Field (mT)

Fig. 1. EPR measurements of irradiated fructose by (A) pulsed EPR and (B) CW-EPR as absorption signals. The inset in B is the first derivative signal of the 100-kGy sample by CW-EPR.

M. Kikuchi et al. / Radiation Physics and Chemistry 81 (2012) 1639–1645

was 200 ns and the recycle delay was 1 ms. In each relaxation measurement, we employed a specific pulse sequence. For T2 determination, we used the two pulse sequence, p/2 pulse–t–p pulse–t–(echo). Each t in the sequence was increased from 200 ns with 8 ns stepwise. In contrast, for T1 determination, we used the following three pulse sequence (i.e., the inversion recovery method): p pulse–T–p/2 pulse–t–p pulse–t–(echo). Here, we changed T from 2000 ns with 400 ns stepwise, fixed t as 200 ns and employed the same parameter sets as those used for the spinecho measurements.

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irradiation, non-irradiated sucrose crystals may exhibit very broad and weak EPR signals. In addition, there is no evidence of a hyperfine structure on these crystals. Therefore, our observation measured by pulsed EPR was different from their CW-EPR signal because the pulsed EPR is specifically sensitive to radicals occurring in close radical pairs.

3.2. Field-swept signal of ESE Fig. 1A shows field-swept echo spectra of radicals in the

g-irradiated fructose by using pulsed EPR. We found that the 3. Results 3.1. Detection of a spin-echo signal after irradiation

Signal Intensity (AU)

Before g-irradiation, no echo signal was detected from the saccharide specimen. We detected the echo signal only after g-irradiation (e.g., 1 kGy). Flores et al. (2000) noted that before

signal spectra depend on the irradiation dose. There are three signals at 1 kGy: the main peak (M1) and two side signals (S1 and S2). There are four signals at 100 kGy: the two main peaks (M1 and M2) and the two side peaks (S1 and S2). Fig. 1B shows the CW-EPR measurements of irradiated fructose. CW spectra showed hyperfine splitting of 5.0 mT between the two side peaks. The field-swept echo yielded a splitting of ca

500

1000

1500

2000

2500

3000 [ns]

0

1000

2000

3000

4000

5000

6000 [ns]

-20

0 [MHz]

Signal Intensity (AU)

0

-60

-40

20

40

60

Fig. 2. Decay of electron spin-echo signal from irradiated fructose: (A) main peak, (B) side peak, and (C) Fourier transformation of the main peak.

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3.4. Hyperfine interaction detected by ESEEM

5 mT as shown in Fig. 1A. Thus, we conclude that both EPR measurements show the same radical species. The M1 and M2 peaks of fructose measured with pulsed EPR split wider after increasing the dose. Therefore, the interaction of M1 and M2 radicals was affected by increasing the dose. This suggests that the two signals are caused by hyperfine splitting.

Fig. 2 shows the decay of an electron spin-echo signal from the irradiated fructose sample. In Fig. 2A, one may immediately notice ESEEM over the echo decay, especially at the initial time range (600 ns). We found a weakly coupled echo modulation near the irradiated radicals of M1 and M2 due to their nutation. Otherwise, we could not detect ESEEM from side peaks in Fig. 2B, indicating that the radicals showing these peaks were not located near the nutation coupling.

3.3. Relaxation times from irradiated fructose We measured the relaxation times (T1 and T2) of the radicals in irradiated fructose based on the pulse echo sequence. The T1 and T2 of the main peak from the 1-kGy-irradiated fructose were 18.5 ms, and 963 ns, respectively. Furthermore, the T1 and T2 of the side peaks from 1-kGy irradiation were 33 ms and about 1470 ns, respectively, in contrast, the T1 and T2 of the main peaks from 100-kGy irradiation were about 24 ms and 140 ns, respectively, and those of the side peaks were around 27 ms and 190 ns, respectively. Comparing the relaxation times of the main and side peaks in each irradiated sample, both T1 and T2 of the side peaks were longer than those of the main peak(s). The difference in relaxation phenomena between the side and main peaks is likely related to the environment surrounding the radicals that show the main or side peaks, i.e., interactions with neighbor electrons and nuclei. With respect to the influence by g-radiation, the T1 of the main and side peaks remained constant by increasing the dose. The T2 of the main and side peaks were drastically reduced. These changes in T2 in response to irradiation indicate that radicals piled up and interacted with the neighbor radicals induced after irradiation.

M1

M2

Signal Intensity (AU)

100 kGy

M1

Fig. 2C indicates the Fourier transformation spectrum of the ESEEM shown in Fig. 2A. Hyperfine interactions indicated by the arrows yielded hyperfine values of 14 and 28 MHz, which are due to proton interactions. The result after Fourier transformation indicates a proton hyperfine effect in the irradiated fructose. The hyperfine effect observed in the main peak constitutes experimental evidence for a C-centered radical. 3.6. Pulsed EPR spectra of mono- and disaccharides Fig. 3 shows the pulsed EPR measurements of irradiated galactose. This hexose had two main peaks and one side peak. The pulsed EPR spectra of irradiated lactose are depicted in Fig. 4. This disaccharide is formed by a b-(1-4) glycoside bond between galactose and glucose. The radicals of lactose showed two main peaks and two side peaks whose signal intensity ratios were no different after increasing the radiation by the

S2

100 kGy

S2

25 kGy

S2

1 kGy

M2

25 kGy

M1

3.5. Fourier transformation of ESEEM

M2

1 kGy

336

344 Magnetic Field (mT)

332

341 Magnetic Field (mT)

Fig. 3. EPR measurements of irradiated galactose by (A) pulsed EPR and (B) CW-EPR as absorption signals. The inset in B is the first derivative signal from the 100-kGy sample by CW-EPR.

M. Kikuchi et al. / Radiation Physics and Chemistry 81 (2012) 1639–1645

100 kGy

M1

M2

Signal Intensity (AU)

S1

1643

100 kGy S2

M1

25 kGy

M2 25 kGy

S1

S2

M1

M2

1 kGy

1 kGy S1

S2

336

344 332 Magnetic Field (mT)

341 Magnetic Field (mT)

Fig. 4. EPR measurements of irradiated lactose by (A) pulsed EPR and (B) CW-EPR as absorption signals. The inset in B is the first derivative signal of the 100-kGy sample by CW-EPR.

pulsed EPR spin-echo method. Both Figs. 3 and 4 show side peaks with lower signal intensities in CW-EPR than in pulsed EPR.

3.7. Dose dependency of relaxation times In Table 1, the relation between relaxation time and irradiation dose was similar among saccharides. T1 is independent of the dose. However, T2 decreases as the dose increases. Furthermore, we could not detect spin-echo signals from non-irradiated saccharides, suggesting that spin-echo is due to irradiation. In our pulsed EPR measurements, the intensities of the echo signals were measured at their peaks following the 24-ns pulse as a p pulse. The pulse length is approximately the same as the ca 41-MHz modulation width, which is equivalent to 1.3 mT. Therefore, the line widths in EPR spectra become broader and lead to overlap of neighboring peaks. Depending on the degree, this overlap may give rise to experimental error, i.e., fluctuation of T1 and T2.

4. Discussion 4.1. Difference between CW and pulsed EPR spectra The difference in peak intensities between CW and pulsed EPR measurements indicates that pulsed EPR spectroscopy by the spin-echo method cannot measure all radicals but only those that closely interact with unpaired electrons. Furthermore, side peaks could show relatively strong EPR spin-echo signals even in 1-kGyirradiated saccharides. Therefore, the interaction between neighboring spins due to specific pairwise trapping of radicals suggests that the pairwise electrons are related to the process of induction.

4.2. Insights into the structure of radicals In the reports of Vanhaelewyn et al. (2001, 2006), single crystals of fructose were investigated at ambient temperature and 60 K by using the angular signal dependency of the crystal axes to clarify the structure of the radical. Pauwels et al. (2002) assigned two radical models based on density functional theory (DFT) to two dominant EIE components distinguished by Vanhaelewyn et al. In a recent report, Tarpan et al. (2010) investigated the radical structure of irradiated fructose precisely by using DFT and EPR/ENDOR/EIE techniques. Sixty-one radical models were calculated for isotropic or anisotropic values and principal directions of the proton hyperfine coupling constant, and compared with six experimentally identified radicals. Two dominant radicals analyzed by Vanhaelewyn et al. (2001) at g¼2.005 were assigned to the C(2)-radical induced by OH abstraction and the C(3)-radical induced after cleavage of the C(2)–C(3) bond rearranged from H abstraction of OH at C(2). In this report, irradiation was applied to the powder form of saccharides at room temperature. The spectrum of fructose powder shown in the inset of Fig. 1B was in good agreement with that reported by Vanhaelewyn et al. (2001). Therefore, the spectra recorded in this experiment were probably from dominant stable radicals in the irradiated saccharide powder because of the irradiation and EPR condition. In our experiments, the g-values of the M1 and M2 peaks were calculated as 2.010 and 2.000, respectively. Therefore, the radicals of M1 and M2 were different from those analyzed by Vanhaelewyn et al. at g¼2.005. The M1 and M2 echo-signals indicated that hydrogen is located near the radical because a nutation of 14 MHz was observed when the T2 decay was recorded. Therefore, M1 and M2 peaks can be said to arise from the C-centered radicals of irradiated fructose after abstraction of the hydroxyl group. Meanwhile, the radicals of the S1 and S2 signals whose g-values were 2.019 and 1.991, respectively, existed without a neighboring hydrogen

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Table 1 Summary of relaxation times T1 and T2 measured in pulsed EPR spectroscopy. Saccharides

Dose (kGy)

T1 (ms) M1

M2

T2 (ns) S1

S2

M1

M2

Glucose

1 10 25 50 100

42.5 33.9 29.9 28.3 27.2

40.2 31.8 30.1 29.4 28.3

817 376 239 179 152

803 343 227 172 148

Maltose

1 10 25 50 100

24.8 23.3 21.2 19.4 17.3

28.7 25.3 21.9 18.9 16.9

486 261 159 135 121

473 245 156 122 117

Fructose

1 10 25 50 100

18.5 18.8 21.9 24.8 23.4

27.3 26.0 24.7

Sucrose

1 10 25 50 100

17.8 18.5 18.0 17.1 15.9

17.6 18.6 18.3 17.7 16.9

Galactose

1 10 25 50 100

49.6 50.3 39.2 34.8 34.9

34.3 34.8 32.9 30.3 30.0

Lactose

1 10 25 50 100

41.5 39.4 36.8 34.3 31.3

41.5 39.6 37.5 35.1 33.5

32.5 35.9 34.3 30.7 28.9

47.2 55.5 44.5 45.0 41.0

32.9 37.8 35.5 32.3 26.0

963 418 273 199 139

293 196 143

402 327 271 199 155

403 328 266 201 150

55.2 62.9 54.3 48.8 45.1

1165 522 277 201 166

850 388 237 176 146

47.1 55.8 49.5 45.1 44.5

563 331 224 176 126

559 327 224 173 138

S1

S2

1564 677 458 316 216

1373 681 446 301 167

1514 627 382 281 219 597 459 340 271 212

576 457 339 262 212

because no nutation was observed. Presumably, the radicals were caused by H abstraction from C atoms or OH groups. The similarity of the relaxation times between M1 and M2, or between S1 and S2 calculated from echo-signals, is attributed to dipole interactions. CW-EPR measurements are recorded from unpaired electrons in irradiated saccharides without disturbing the spin system of the samples. Based on the measurement principles, pulsed EPR spectroscopy with the spin-echo method should be capable of measuring strong interactions between the radical pairs within the inter-spin distance of ca 1 nm (or somewhat more). Therefore, pulsed EPR focuses on physically interacting radicals as a specific component of CW-EPR measurements. 4.3. T1 as a function of irradiation dose A relationship between the relaxation time and the irradiation dose is observed. In principle, the relaxation time T1 relates to the energy transfer through interactions between the electron spin and the lattice. Upon an increase of irradiation, T1 was hardly changed by the spin concentration.

correlated radical pairs with rather short inter-spin distance (ca 1 nm or so), which exist at room temperature. Previously such effect was found for radiolysis of organic crystals at low temperatures (Iwasaki and Ichikawa, 1967; Gillbro et al., 1969; Gillbro and Lund, 1974, 1976; Nilsson and Lund, 1984). On the other hand, the dose-dependency of T2 in the irradiated saccharide indicates that a shorter T2 is induced by stronger spin–spin interactions because of free radicals induced by radiation. This interaction is related to the inter-electron distance. The increase in the unpaired electron content (formation of random or noncorrelated pairs) after g-irradiation causes stronger interactions that induce a shorter T2. In the case of dominating contribution of correlated radical pairs over the whole dose range, T2 would be almost independent of dose since it is determined only by local concentration of radicals (Gillbro and Lund, 1974, 1976). Apparently, it is not the case in the present work. Moreover, these responses may be affected by differences in the environment of the radicals in saccharide. The T2 values of the side peaks were longer than those of the main peaks in galactose and fructose, indicating that radicals of the side peaks interact less with intramolecular dipoles than those of the main peaks. The technique of relaxation time analyses by the spin-echo technique of pulsed EPR might be able to distinguish irradiated foods by the EPR signals of radiation-induced radicals among all radicals. Initially, the finding of a spin-echo signal by pulsed EPR measurement may show the irradiation history of the samples. Subsequently, the evaluation of relaxation time T2 from stable radicals in saccharide enables one to know the g-irradiation dose according to the dose-dependency of T2.

4.5. Practical applications EPR spectroscopy has been utilized to investigate photochemical processes after irradiation (Shimada, 1992). High-energy radiation ionizes matter directly by scattering electrons and/or excites atoms (molecules) by transferring energy (Christophorou, 1971). The excited atoms (molecules) release the energy to the ground state or cleave chemical bonds in polymers (Makhlis, 1975). The cleaved molecule can be detected by EPR spectroscopy when the induced unpaired electrons have long lifetimes. The radiation-induced cleavages in crystals show radical interactions because unpaired electrons may exist in neighbors by motional restriction. To investigate an interaction between radicals, the relaxation times of T1 and T2 are comparable indicators. The relaxation times have usually been calculated by using a simulation technique from signal saturation behaviors (Lund et al., 2009) because direct measurement with pulsed EPR was generally observable within a limited range of T1 and T2 values. In this report, a parameter applicable to a disordered system of saccharides is found to measure the relaxation times directly without the large discrepancies that occasionally come from the applicability limits of a simulation technique. Therefore, despite radical induction in foods during powdering treatment, pulsed EPR spectroscopy by spin-echo detection may be advantageous for detecting radiation-induced radicals.

4.4. T2 as a function of irradiation dose Karakirova et al. (2010) showed that line-width (by CW-EPR) increased with the irradiation dose in saccharides. This indicates that T2 decreases as the dose increases. In fact, we directly determined the line-width increment experimentally by pulse EPR spectroscopy. The relaxation time T2 relates to the interaction between the spins. The results shown in this paper suggest that, even at low doses, the radicals are trapped predominantly in relatively close

5. Conclusions The basic conclusions can be summarized as follows (i) the spin–lattice relaxation time (T1) is virtually independent of the absorbed dose, and (ii) the spin–spin relaxation time (T2) decreases as the dose increases, reflecting the increasing concentration of radicals. EPR spectroscopy by spin-echo detection is a useful technique to evaluate radical interactions.

M. Kikuchi et al. / Radiation Physics and Chemistry 81 (2012) 1639–1645

Acknowledgment We are grateful for the ‘‘Basic research on utility of radiation for foods and its practical application’’ Grant under the Strategic Promotion Program (2008–2010) for Basic Nuclear Research by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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