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Preparation and EPR Studies of Lithium Phthalocyanine Radical as an Oxymetric Probe
Free Radical Biology & Medicine, Vol. 25, No. 1, pp. 72–78, 1998 Published by Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
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Original Contribution PREPARATION AND EPR STUDIES OF LITHIUM PHTHALOCYANINE RADICAL AS AN OXYMETRIC PROBE MOBAE AFEWORKI, NATHAN R. MILLER, NALLATHAMBY DEVASAHAYAM, JOHN COOK, JAMES B. MITCHELL, S. SUBRAMANIAN, and MURALI C. KRISHNA Radiation Biology Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA (Received 14 November 1997; Revised 23 January 1998; Accepted 27 January 1998)
Abstract—The electron paramagnetic resonance (EPR) spectrum of the paramagnetic center in solid lithium phthalocyanine, LiPc, exhibits a pO2 (partial pressure of oxygen)-dependent line width. The compound is insoluble in water and is not easily biodegradable and, therefore, is a useful spin probe for quantitative in vivo oxymetry. Because EPR spectrometry is potentially a useful technique to quantitatively obtain in vivo tissue pO2, such probes can be used to obtain physiological information. In this paper, a simple experimental procedure for the preparation of LiPc using potentiostatic electrochemical methods is described. The setup was relatively inexpensive and easy to implement. A constant potential ranging from 0.05 to 0.75 V versus Ag1/AgCl(s) was used for obtaining LiPc. The EPR spectral studies were carried out using spectrometers operating at X-band and at radiofrequency (RF) at different pO2 values to characterize the spectral response of these crystals. The results indicate that, depending on the electrolysis conditions, the products contain mixtures of crystals exhibiting pO2-sensitive and pO2-insensitive line widths. Electrolysis conditions are reported whereby the pO2-sensitive LiPc crystals were the predominant product. The influence of the working surface of the electrode and the electrolysis time on the yield were also evaluated. The crystals of LiPc were also studied using a time-domain RF EPR spectrometer. In time-domain EPR, the signals that survive beyond the spectrometer dead time are mainly the narrow lines corresponding to the pO2-sensitive crystals, whereas the signals arising from the pO2-insensitive component of LiPc were found not to survive beyond the spectrometer dead time. This signal survival makes the time-domain EPR method more sensitive for pO2 measurements using LiPc because the line width becomes very narrow at very low pO2 and, concomitantly, the relaxation time T 2 longer, with no modulation or power saturation artifacts that are encountered as in the continuous wave (cw) mode. Further, minimal contributions from object motion in the spectral data obtained using time-domain methods make it an advantage for in vivo applications. Published by Elsevier Science Inc. Keywords—Lithium phthalocyanine, Oxymetry, FT EPR, RF EPR, Free radical INTRODUCTION
tion. The effect of dissolved oxygen in broadening the EPR spectral line widths of suitable spin probes forms the basis of oxymetry using EPR spectroscopy.2–5 Earlier workers6 –9 have demonstrated the use of such effect to measure concentration of dissolved oxygen in various chemical and biological studies including in vivo objects using spin probes such as stable nitroxide free radicals,10 and particulate probes such as fusinite,11 glucose char,12 India ink,13 and LiPc.8 Infusible spin probes such as nitroxides have been found to be useful in obtaining spatial information on oxygen distribution using spectral-spatial imaging techniques,7,9 whereas particulate spin probes such as fusinite, glucose char and LiPc have been useful in making repeated in vivo measurements. In addition, EPR methods similar to oxymetry have also been used to estimate NO concentrations based on the
In vivo oxymetric methods are being evaluated for use in humans to guide therapeutic regimens for the effective treatment of solid malignancies containing zones of hypoxia.1 Useful methods to determine oxygen concentration in vivo should be quantitative and noninvasive, and performing repeated measurements as a function of treatment course should be possible. In addition, because measuring hypoxia is more important, the oxymetric method should have increased sensitivity at low oxygen concentrations. Though several methods exist to measure tissue pO2,1 EPR methods are receiving increasing attenAddress correspondence to: Dr. Murali C. Krishna, Radiation Biology Branch/NCI, Bldg. 10, Room B3-B69, Bethesda, MD 20892; Tel: (301) 496-7511; Fax: 301-480-2238; E-Mail: [email protected]. 72
Lithium phthalocyanine: synthesis and oxymetry
same physical principles.14 Though nitroxide spin probes have been found to have linear response to oxygen,15 at low pO2 (,10 mmHg) LiPc was found to be more desirable for oxymetric applications because of the narrow spectral line widths.8 The very narrow lines found in oxygen free LiPc have been attributed to exchange narrowing in the lattice which exhibit a pO2-dependent line broadening due to dipolar coupling of the paramagnetic center with triplet state of the ground state O2 molecules16 conferring the necessary physical properties for use in oxymetry. The procedures for the preparation of most of the spin probes mentioned above (such as mH-CTPO, fusinite, glucose char) have been available whereas others are naturally available (such as India ink, fusinite). Most of these agents exhibit EPR line widths in the range of several tens of milligauss to several gauss between 0% and 100% pO2 conditions. Oxymetry with nitroxides, though linear in response over a wide range of pO2, is difficult to carry out when the pO2 range is very low; whereas with LiPc, oxymetry is possible even at low pO2 levels.17 Therefore, LiPc is more useful as an oxymetric probe at clinically relevant pathological conditions such as those encountered in solid tumors with compromised blood supply. LiPc and other spin probes have been implanted in experimental animal models and repeated and quantitative oxygen measurements were performed in vivo.8,17,18 These studies supported the utility of EPR oxymetry for repeated pO2 measurements in preclinical experimental models of cancer treatment. However, further progress in this direction has been limited because of lack of commercial availability of pO2-sensitive LiPc suitable for in vivo oxymetric applications. This study reports the detailed procedure for the electrochemical synthesis of LiPc that is fairly simple and easy to implement. The spectral properties at different pO2 values were characterized by continuous wave (cw) and time domain EPR spectroscopy. MATERIALS AND METHODS
Apparatus and chemicals The dilithium salt of phthalocyanine, Li2Pc, was purchased from Aldrich Chemical Co. (Milwaukee, WI); the Ag1/AgCl(s) reference electrode and the CV-27 Voltammograph from Bioanalytical Systems, Inc. (West Lafayette, IN); HPLC-grade tetraethyl ammonium perchlorate (TEAP) from Fluka Chemie (Switzerland) and acetonitrile (water content of 10 ppm) from Burdick & Jackson, Inc. (Muskegon, MI); phosphate-buffered solution (PBS) at pH 7.4, without calcium and without magnesium, from Biofluids, Inc. (Rockville, MD); and gaspermeable teflon capillary tube of 0.8 mm inner diameter
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Fig. 1. An electrochemical cell for the potentiostatic preparation of LiPc using a Pt gauze as the working electrode (W). The working electrodes used were a Pt gauze, Pt coiled-coil, and Pt coil; the details of these electrodes are given in the text. The reference electrode (R) was a Ag1/AgCl(s) and the auxiliary electrode (A) was a Pt wire. The separation between the electrodes X, Y, and Z were about 18, 13, and 13 mm, respectively.
and 0.025 mm wall thickness from Zeus Industrial Products, Inc. (Orangeburg, SC). The electrochemical cell shown in Fig. 1 uses platinum electrodes for both the working and auxiliary electrodes. The auxiliary electrode was a Pt wire, while the working electrode was either a Pt gauze (450 squares/cm2 with a wire thickness of 0.20 mm (32 ga.); 40 mm diameter and 50 mm length), a coiled Pt wire (with a wire size of 0.63 mm (22 ga.); coil i.d. 0.8 mm and length 30 mm), or a helical coiled coil (with a wire size of 0.63 mm (22 ga.); small coil i.d. 0.8 mm, large coil i.d. 10 mm, length 60 mm). No special surface treatment was given to the platinum electrodes; and all chemicals were used as obtained from the manufacturers. Electrochemical synthesis of LiPc LiPc was synthesized by a procedure similar to those reported earlier19,20 with minor modifications: the elec-
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trosynthesis was performed under air and at constant potential resulting in pO2-sensitive crystals. The dilithium salt Li2Pc was oxidized to LiPc in acetonitrile using TEAP as the supporting electrolyte, at a controlled potential using the CV-27 Voltammograph in an electrochemical cell (a 100-ml glass cell vial) using Pt electrodes. Several batches of LiPc were prepared at an oxidation potential that was maintained at a given voltage, from 0.05 to 0.75 V versus Ag1/AgCl(s) electrode. Platinum electrodes were used for both the working and auxiliary electrodes. The auxiliary electrode was a coiled Pt wire inside a fritted glass, while the working electrode was either a Pt gauze, a Pt coil, or a Pt coiled coil. To a 70-ml volume of acetonitrile containing 0.1 M TEAP, 200 mg of Li2Pc were added with constant stirring to obtain a dark blue solution. The stirring continued for about 1 h to completely dissolve the Li2Pc. The cell was kept at room temperature (no special efforts were undertaken to thermostat the vessel) and the electrolysis initiated at constant potential and continued till the completion of the reaction, where the intensity of the electrolysis current was about the same as the residual current. The contents of the cell were not stirred but were covered during the electrochemical oxidation process to avoid dirt and reduce evaporation rate. The completion of the reaction took several days at the lower potential and hours at the higher potentials, 6 –7 days at 0.05 V and about 1 h at 0.75 V. Crystals of LiPc were deposited onto the electrode surface or dropped out of solution as LiPc is not soluble in acetonitrile. As expected, for the same potential, the working electrode with the largest surface area led to the fastest reaction. The LiPc crystals thus obtained were filtered, washed several times with 10-ml portions of acetonitrile, and dried at 85°C overnight. EPR sample preparation Quantitative measurements of the dependence of the EPR line width of LiPc as a function of pO2 were carried out. LiPc samples kept in vials covered with air-tight septa were equilibrated with O2/N2 mixtures of various compositions using hypodermic syringe needles as inlet and outlet. For RF EPR measurements the resonator sizes allowed the use of 15-mm diameter vials and the LiPc samples in the vials were loaded into the resonator directly. For X-band EPR measurements, a suspension of LiPc in PBS (100 ml) was drawn into a gas-permeable, flexible Teflon capillary tube. The tube was folded twice so that the sample occupies the active volume of the cavity. The folded tube was inserted into a 4-mm quartz tube open at both ends and was inserted into the rectangular EPR cavity configured to remain horizontal. The quartz tube containing the sample was slowly purged
with O2/N2 mixture of a desired composition at one end. Equilibration of the system with the available O2 whether at RF or X-band frequencies was ascertained by making sure that the amplitudes reach a constant value. For our experimental set up, this was achieved within a few minutes, but purging with the gas continued for 20 min and throughout the measurement time.
EPR measurements X-band cw EPR measurements were carried out using a Varian E9 spectrometer with TE102 microwave cavity, at a nonsaturating microwave power of 0.2 mW and a field of 3375 G. Due to the inhomogeneity of the magnetic field of the X-band EPR spectrometer over the sample volume, the spectra were deliberately over modulated to broaden out the spectral splittings caused by the inadvertent spatial encoding. The RF (289 MHz) cw EPR measurements were made on a home-built spectrometer, using a 15-mm helical coil resonator with 3.13 kHz field modulation and phase-sensitive detection. The time-domain EPR measurements were obtained using a home built RF (300 MHz) EPR spectrometer, described elsewhere,21,22 that uses a 1-GHz sampler with a channel limitation of 4 k points, corresponding to 4.096 ms. Peak heights reported are peak-to-peak of the first derivative of the absorption mode in the case of both of the RF and X-band cw EPR experiments; the line width in the pulsed RF EPR experiments is the full width at half maximum (FWHM) of the unmodulated absorption line. RESULTS
Several batches of LiPc crystals synthesized at different oxidation potentials were studied for their line widths and oxygen response using three different EPR spectrometers: a cw X-band (9.3 GHz), a cw RF (289 MHz), and a time-domain RF (300 MHz). The response of the line widths of LiPc crystals to oxygen was studied from 0% pO2 (obtained by purging with N2 gas) to 100% pO2 (obtained by purging with O2 gas) condition. A typical response of the line width of an LiPc batch that was found to be pO2-sensitive is shown in Fig. 2. This LiPc batch, synthesized at a constant potential of 0.35 V using a Pt gauze working electrode, was almost exclusively composed of crystals whose line width was sensitive to pO2, the line width ranging from 50 mG to 1.3 G from 0% to 100% O2, respectively. However, an LiPc batch, synthesized at a constant potential of 0.15 V using a coiled-coil working electrode, gave mainly a broad line with a line width of 300 mG which was pO2-independent. In addition, a minor component with a narrow line whose line width and amplitude depend on pO2 was
Lithium phthalocyanine: synthesis and oxymetry
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Fig. 2. Continuous wave (cw) X-band EPR spectra of an oxygen-dependent LiPc batch (electrochemically synthesized at a constant potential of 0.35 using a Pt gauze working electrode) under different pO2 levels. The oxygen percentages from the narrowest to the broadest spectra were: 0.10%, 0.21%, 2.04%, 5.02%, 21%, and 100%, and the corresponding receiver gains were 1.25 3 103, 1.25 3 103, 2.5 3 103, 4 3 103, and 2.5 3 104, respectively. The spectrometer settings were: modulation amplitude, MA, 0.8 G; modulation frequency, MF, 25 kHz; microwave power, P, 0.2 mW; sweep width, SW, 20 G; time constant, TC, 1 s; scan time, ST, 4 min. The spectra were severely overmodulated to avoid multiplets caused due to magnet inhomogeneity, and thus were used only in broad qualitative terms. The inset shows the spectra of an LiPc batch (electrochemically synthesized at a constant potential of 0.15 V using a coiled coil working electrode) that is predominantly composed of oxygen-insensitive crystals of LiPc at 0% O2 and 21% O2 (compressed air); the broad and narrow lines correspond to the oxygen-insensitive and oxygen-sensitive crystals, respectively.
observed. The inset in Fig. 2 shows a typical EPR spectra of an LiPc batch that partly responds to oxygen, under two different pO2s, 0% O2 and air (about 21% O2), respectively. The LiPc crystals, synthesized by electrolysis at 0.35 V that exhibited predominantly pO2-dependent EPR line widths, were examined for their spectral characteristics at different oxygen levels by EPR spectroscopy at RF and X-band frequencies and the results are shown in Fig. 3. The line width of the LiPc crystals were found to linearly increase as a function of pO2. The inverse of the amplitude, which is more sensitive to changes in pO2, also increases with pO2 and is shown in Fig. 3, for both the X-band and RF cw EPR measurements. The homogeneity of the magnet of the X-band EPR spectrometer was not sufficient enough to reliably measure the line widths of the paramagnetic center in LiPc at low pO2 with expected line widths of less than 20 mG. In fact, undesired line splitting was observed due to the spatial encoding of the LiPc particles due to the inhomogeneity of the magnetic field. To estimate the dependence of the spectra on pO2, we deliberately overmodulated the spectral lines with modulation amplitudes higher than the line width to get an envelope of the spectra, and then used only the amplitudes of these broadened lines to correlate to the pO2. The amplitude of the lines correlated inversely to the pO2. However, these
data were not utilized to measure any relaxation times or to evaluate broadening mechanisms. In time-domain EPR methods, saturation effects and modulation artifacts on the spectral line widths are avoided. Therefore, the same batches of LiPc were also studied using a pulsed RF FT EPR spectrometer operat-
Fig. 3. The inverse amplitude (peak-to-peak height) as a function of percent oxygen from the X-band continuous wave (cw) and the RF cw EPR spectrometers (with spectrometer settings: MA 0.5; MF 3.13 kHz; P 2 13 dBm; SW 20 G; TC 640 ms; ST 500 s). The oxygen-sensitive batch shows an amplitude and line width that strongly depend on pO2. The inverse of the peak heights of the first derivative EPR signal versus percent oxygen for both the cw X-band and cw RF EPR cases show a linear increase with pO2.
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Fig. 4. The FT spectra of the oxygen-sensitive LiPc batch shown in Fig. 2, using a time-domain RF EPR spectrometer at two different oxygen percentages, 0% O2 and 2.04% O2, respectively. A 1-GHz sampler with a channel limitation of 4 k points, corresponding to 4.096 ms was used to collect the data. The wiggles in the 0% O2 data are due to truncation of the FID at 4.1 ms, no digital filters were used.
ing at 300 MHz. Similar to the cw EPR measurements, the crystals show pO2-dependent line widths and amplitudes. Typical FT spectra are shown in Fig. 4 at two different pO2s of 0% O2 and 2.04% O2 for the same batch whose cw spectra and its oxygen response are shown in Figs. 2 and 3, respectively. The pO2 dependence of the line width of the FT spectra obtained with the pulsed EPR spectrometer is shown in Fig. 5. The line shape of the spectrum obtained from LiPc crystals at low pO2 using time-domain RF EPR spectrometer were fitted to a single exponential decay constant. The line width of the FT spectra increases with increasing pO2. The line
Fig. 5. A plot of the full width at half maximum in kilohertz versus percent oxygen. The line width under nitrogen atmosphere was 350 kHz and increases linearly, with a sensitivity of 195 kHz (70 mG) per percent oxygen, R 2 5 0.996, as the percent oxygen is increased reaching 2400 kHz at 7.5%. Above 7.5% O2, the signal was still measurable but the spectra obtained were so noisy that determining the line widths from them was not even attempted.
widths of the spectra from the time-domain RF EPR data were plotted versus percent oxygen and fitted to a straight line, which gave a sensitivity of 195 kHz (70 mG) per percent oxygen (R2 5 0.996). As the pO2 was increased, the free induction decay (FID) survives for shorter time intervals (the line width increases), decreasing the amplitude of the detected signal substantially because an appreciable fraction of the signal was lost within the spectrometer dead time. As the pO2 is increased to 7.5% and higher, the signal-to-noise was prohibitively low to do accurate line width measurements. Batches of LiPc whose spectral properties did not show strong pO2 dependence, in which the broad (300 mG) pO2-independent line is the dominant component of the product in the cw measurements, do show pO2 dependence in the pulsed mode. Whereas the cw measurements detect both the broad and narrow lines, the pulsed mode more favorably detects narrow lines because the FIDs corresponding to narrow lines persist beyond the spectrometer dead time, while those corresponding to broad lines decay completely within the dead time. Thus, whereas the cw measurements show the narrow lines as peaks on top of the broad lines, the pulsed EPR measurements show the narrow lines almost exclusively. The dependence of the electrolytic conditions on the yield of the LiPc crystals and their spectral properties was investigated by varying the electrode geometry as well as the electrolysis potential. The variation of the Pt working electrode from a gauze, to a coil, to a coiled coil gave different types of crystals with respect to their response to pO2. The Pt gauze gave pO2-sensitive crystals of LiPc over a broad range of potential. Oxygensensitive LiPc was obtained at all potentials used (0.05– 0.55 V) to varying fraction of the total product. Electrolysis in the range of 0.25– 0.35 V gave crystals that were predominantly pO2-sensitive, whereas the lower potentials gave crystals that were predominantly pO2-insensitive. Electrolysis with the helical coiled coil, on the other hand, gave pO2-sensitive crystals at potentials above 0.25 V; at potentials below 0.25 V the crystals obtained were almost exclusively pO2-insensitive, while those synthesized above 0.35 V were predominantly pO2-sensitive. The crystals obtained using the coiled coil were larger compared with those obtained by using a gauze. Electrolysis with the simple helical coil gave pO2-sensitive crystals at potentials above 0.35 V. Here again, at 0.25 V and below the crystals obtained were exclusively pO2-insensitive. The size of the crystals obtained using the simple coil were similar to those obtained using helical coiled-coil. Attempts to synthesize the LiPc using the same set up as above, but in a constant current mode at 5, 20, and 50 mA for 24, 48, and 72 h, respectively, produced LiPc crystals but failed to produce pO2-sensitive crystals. At-
Lithium phthalocyanine: synthesis and oxymetry
tempts were also made to see whether the pO2-sensitive crystals can be separated from the mixture of products containing both pO2-sensitive and pO2-insensitive batches by recrystallization from a suitable solvent such as chloronaphthalene. But such attempts were not successful in obtaining useful product for oxymetry, because the recrystallized products exhibited EPR spectra whose line widths were not pO2-dependent. DISCUSSION
The electrochemical synthetic route for the preparation of LiPc described in this paper shows the feasibility of obtaining useful quality particulate spin probes for oxymetry in a relatively simple and inexpensive manner. The oxidation of Li2Pc to LiPc has been found to be a straight forward reaction dependent mainly on the surface area of the working electrode and the potential applied. The Pt gauze with the largest surface area leads to shorter oxidation times at a given potential because the LiPc crystals formed are deposited on the surface of the electrode and the Pt gauze has the largest surface area. The crystals formed can be classified into two types based on the response of their EPR line width to pO2. For all three working electrodes used, those batches that were synthesized at lower potentials (0.05– 0.15 V) were predominantly, and in some cases almost exclusively, the pO2-insensitive crystals; batches that were synthesized at intermediate potentials (0.25– 0.35 V) gave mainly the pO2-sensitive crystals, while those synthesized above 0.35 V gave mixtures of the two types, with increasing proportion of the pO2-insensitive crystals as the potential is raised. At 0.75 V, using a coiled coil, the product was mainly pO2-insensitive. Contrary to reports in the literature, which recommend electrolysis at constant current,23 the results from this study indicate that pO2sensitive LiPc can be synthesized potentiostatically. Crystal structure data of the lithium complex of phthalocyanine radicals reported in the literature give two different forms: a tetragonal of space group P4/nnc, with a 5 b 5 19.6 Å, c 5 6.4 Å 19,23 and a tetragonal of space group P4/mcc, with a 5 b 5 13.8 Å, c 5 6.403 Å. 23 The former structure gives a pO2-independent EPR signal (300 mG) while the latter structure gives a pO2dependent EPR signal (20 mG to 1.3 G). Based on the reported space groups, the pO2-sensitive batches we prepared are consistent with the P4/mcc space group, whereas those batches that give pO2-independent EPR signal are consistent with the P4/nnc space group. The line width of the pO2-sensitive LiPc, unlike many other carbon-based paramagnetic materials, and spin probes such as triacetoneaminoxyl (TANO)23 and other nitroxides, increases linearly as a function of pO2 from 0% to 100% pO2. In the absence of oxygen, the line
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width of the pO2-sensitive LiPc was estimated to be approximately 50 mG. This width is broader than the reported line width of 14-20 mG8,16,23 due to the inhomogeneity of the magnetic field of the X-band EPR spectrometer. Because of the inhomogeneity of the Xband magnet used in the current study, attempts were not made to measure line widths as a function of pO2. The EPR spectra were overmodulated and amplitudes were used to study the spectral response to pO2. Other workers24 have presented detailed instrumental considerations at X-band to reliably measure the linewidth of LiPc at very low pO2. All batches of LiPc synthesized by the above potentiostatic method were found to be stable at room temperature for periods of several months, the period they were studied, making them useful probes for oxymetry, an advantage compared to other probes such as fluoranthene hexaflourophosphate which are not quite stable at ambient temperatures. LiPc can also be used as a standard in low frequency pulsed EPR systems because it has a long spin-spin relaxation time constant (T 2 ) relative to the spectrometer dead time; it has an FID that lasts for several microseconds that persist beyond the dead time at such frequencies that are of the order of 500 ns. The line shape of the spectrum obtained from LiPc crystals that are subject to minimum pO2 were fitted to a single exponential decay constant corresponding to a one-dimensional linear Heisenberg magnetic exchange. Any attempt to modify the lattice would destroy the exchange coupling resulting in much broader lines that are probably not sensitive to pO2. From the nature of the results obtained from the EPR and electrolysis experiments, it is possible to draw some broad, albeit useful, conclusions. The grain size of the LiPc crystals was found to depend on the actual geometry/shape of the Pt electrode used, namely gauze, coil, coiled-coil. A pO2-sensitive LiPc was produced for the broad range of working potential from 0.10 to 0.75 V in different proportions. The range of potential used also decided the net yield of pO2-sensitive LiPc. The optimal range of potential that gave maximum percentage yield of pO2-sensitive LiPc was 0.25– 0.35 V using the Pt gauze electrode. At potentials lower than 0.25 V the product was mainly pO2-insensitive; at potentials above 0.35 V the percentage of the pO2-sensitive fraction was reduced. Electrochemical procedures described in this report should be useful to prepare these probes with relative ease and good yield making oxymetric studies widely accessible. Further, the EPR methods described to characterize the products for oxymetric applications will help in choosing appropriate experimental conditions to obtain LiPc crystals with pO2-sensitive paramagnetic centers.
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H. M. In vivo oxymetry using EPR and India ink. Magn. Reson. Med. 33:237–245; 1995. Singh, R. J.; Hogg, N.; Mchaourab, H. S.; Kalyanaraman, B. Physical and chemical interactions between nitric oxide and nitroxides. Biochim. Biophys. Acta. 1201:437– 441; 1994. Halpern, H.; Peric, M.; Nguyen, T.-D.; Spencer, D. P. Selective isotopic labeling of a nitroxide spin label to enhance sensitivity for T 2 oxymetry. J. Magn. Reson. 90:40 –51; 1990. Bensebaa, F.; Andre, J.-J. Effect of oxygen on phthalocyanine radicals. 1. ESR study of lithium phthalocyanine spin species at different oxygen concentrations. J. Phys. Chem. 96:5739–5745; 1992. Swartz, H. M.; Boyer, S.; Gast, P.; Glockner, J. F.; Hu, H.; Liu, K. J.; Moussavi, M.; Norby, S. W.; Vahidi, N.; Walczak, T.; Wu, M.; Clarkson, R. B. Measurements of pertinent concentrations of oxygen in vivo. Magn. Res. Med. 20:333–339; 1991. Goda, F.; Ohara, J. A.; Rhodes, E. S.; Liu, K. J.; Dunn, J. F.; Bacic, G.; Swartz, H. M. Changes in oxygen tension in experimental tumors after single dose of x-ray irradiation. Cancer Res. 55: 2249 –2252; 1995. Sugimoto, H.; Mori, M.; Masuda, H.; Taga, T. Synthesis and molecular structure of a lithium complex of the phalocyanine radical. J. Chem. Soc., Chem. Commun. 962–963; 1986. Turek, P.; Andre, J.-J.; Giraudeau, A.; Simon, J. Preparation and study of a lithium phthalocyanine radical: Optical and magnetic properties. Chem. Phys. Lett. 134:471– 476; 1987. Murugesan, R.; Cook, J. A.; Devasahayam, N.; Afeworki, M.; Subramanian, S.; Tschudin, R.; Larsen, J. H.; Mitchell, J. B.; Russo, A.; Krishna, M. C. In vivo imaging of a stable paramagnetic probe by pulsed radiofrequency electron paramagnetic resonance spectroscopy. Magn. Reson. Med. 38:409 – 414; 1997. Murugesan, R.; Afeworki, M.; Cook, J. A.; Devasahayam, N.; Tschudin, R.; Mitchell, J. B.; Subramanian, S.; Krishna, M. C. A broad band pulsed radio-frequency EPR spectrometer for biological applications. Rev. Sci. Instr. 69:1869 –1876; 1998. Moussavi, M. Radical lithium phthalocyanine crystals, their preparation process and their use for the In vivo determination of molecular oxygen. U.S. Patent No. 5,112,597; Date of Patent: May 12, 1992. Smirnov, A. I.; Norby, S.-W.; Walczak, T.; Lui, K. J.; Swartz, H. M. Physical and instrumental considerations in the use of lithium phthalocyanine for measurements of the concentration of oxygen. J. Magn. Reson. B. 103:95–102; 1994.
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Original Contribution PREPARATION AND EPR STUDIES OF LITHIUM PHTHALOCYANINE RADICAL AS AN OXYMETRIC PROBE MOBAE AFEWORKI, NATHAN R. MILLER, NALLATHAMBY DEVASAHAYAM, JOHN COOK, JAMES B. MITCHELL, S. SUBRAMANIAN, and MURALI C. KRISHNA Radiation Biology Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA (Received 14 November 1997; Revised 23 January 1998; Accepted 27 January 1998)
Abstract—The electron paramagnetic resonance (EPR) spectrum of the paramagnetic center in solid lithium phthalocyanine, LiPc, exhibits a pO2 (partial pressure of oxygen)-dependent line width. The compound is insoluble in water and is not easily biodegradable and, therefore, is a useful spin probe for quantitative in vivo oxymetry. Because EPR spectrometry is potentially a useful technique to quantitatively obtain in vivo tissue pO2, such probes can be used to obtain physiological information. In this paper, a simple experimental procedure for the preparation of LiPc using potentiostatic electrochemical methods is described. The setup was relatively inexpensive and easy to implement. A constant potential ranging from 0.05 to 0.75 V versus Ag1/AgCl(s) was used for obtaining LiPc. The EPR spectral studies were carried out using spectrometers operating at X-band and at radiofrequency (RF) at different pO2 values to characterize the spectral response of these crystals. The results indicate that, depending on the electrolysis conditions, the products contain mixtures of crystals exhibiting pO2-sensitive and pO2-insensitive line widths. Electrolysis conditions are reported whereby the pO2-sensitive LiPc crystals were the predominant product. The influence of the working surface of the electrode and the electrolysis time on the yield were also evaluated. The crystals of LiPc were also studied using a time-domain RF EPR spectrometer. In time-domain EPR, the signals that survive beyond the spectrometer dead time are mainly the narrow lines corresponding to the pO2-sensitive crystals, whereas the signals arising from the pO2-insensitive component of LiPc were found not to survive beyond the spectrometer dead time. This signal survival makes the time-domain EPR method more sensitive for pO2 measurements using LiPc because the line width becomes very narrow at very low pO2 and, concomitantly, the relaxation time T 2 longer, with no modulation or power saturation artifacts that are encountered as in the continuous wave (cw) mode. Further, minimal contributions from object motion in the spectral data obtained using time-domain methods make it an advantage for in vivo applications. Published by Elsevier Science Inc. Keywords—Lithium phthalocyanine, Oxymetry, FT EPR, RF EPR, Free radical INTRODUCTION
tion. The effect of dissolved oxygen in broadening the EPR spectral line widths of suitable spin probes forms the basis of oxymetry using EPR spectroscopy.2–5 Earlier workers6 –9 have demonstrated the use of such effect to measure concentration of dissolved oxygen in various chemical and biological studies including in vivo objects using spin probes such as stable nitroxide free radicals,10 and particulate probes such as fusinite,11 glucose char,12 India ink,13 and LiPc.8 Infusible spin probes such as nitroxides have been found to be useful in obtaining spatial information on oxygen distribution using spectral-spatial imaging techniques,7,9 whereas particulate spin probes such as fusinite, glucose char and LiPc have been useful in making repeated in vivo measurements. In addition, EPR methods similar to oxymetry have also been used to estimate NO concentrations based on the
In vivo oxymetric methods are being evaluated for use in humans to guide therapeutic regimens for the effective treatment of solid malignancies containing zones of hypoxia.1 Useful methods to determine oxygen concentration in vivo should be quantitative and noninvasive, and performing repeated measurements as a function of treatment course should be possible. In addition, because measuring hypoxia is more important, the oxymetric method should have increased sensitivity at low oxygen concentrations. Though several methods exist to measure tissue pO2,1 EPR methods are receiving increasing attenAddress correspondence to: Dr. Murali C. Krishna, Radiation Biology Branch/NCI, Bldg. 10, Room B3-B69, Bethesda, MD 20892; Tel: (301) 496-7511; Fax: 301-480-2238; E-Mail: [email protected]. 72
Lithium phthalocyanine: synthesis and oxymetry
same physical principles.14 Though nitroxide spin probes have been found to have linear response to oxygen,15 at low pO2 (,10 mmHg) LiPc was found to be more desirable for oxymetric applications because of the narrow spectral line widths.8 The very narrow lines found in oxygen free LiPc have been attributed to exchange narrowing in the lattice which exhibit a pO2-dependent line broadening due to dipolar coupling of the paramagnetic center with triplet state of the ground state O2 molecules16 conferring the necessary physical properties for use in oxymetry. The procedures for the preparation of most of the spin probes mentioned above (such as mH-CTPO, fusinite, glucose char) have been available whereas others are naturally available (such as India ink, fusinite). Most of these agents exhibit EPR line widths in the range of several tens of milligauss to several gauss between 0% and 100% pO2 conditions. Oxymetry with nitroxides, though linear in response over a wide range of pO2, is difficult to carry out when the pO2 range is very low; whereas with LiPc, oxymetry is possible even at low pO2 levels.17 Therefore, LiPc is more useful as an oxymetric probe at clinically relevant pathological conditions such as those encountered in solid tumors with compromised blood supply. LiPc and other spin probes have been implanted in experimental animal models and repeated and quantitative oxygen measurements were performed in vivo.8,17,18 These studies supported the utility of EPR oxymetry for repeated pO2 measurements in preclinical experimental models of cancer treatment. However, further progress in this direction has been limited because of lack of commercial availability of pO2-sensitive LiPc suitable for in vivo oxymetric applications. This study reports the detailed procedure for the electrochemical synthesis of LiPc that is fairly simple and easy to implement. The spectral properties at different pO2 values were characterized by continuous wave (cw) and time domain EPR spectroscopy. MATERIALS AND METHODS
Apparatus and chemicals The dilithium salt of phthalocyanine, Li2Pc, was purchased from Aldrich Chemical Co. (Milwaukee, WI); the Ag1/AgCl(s) reference electrode and the CV-27 Voltammograph from Bioanalytical Systems, Inc. (West Lafayette, IN); HPLC-grade tetraethyl ammonium perchlorate (TEAP) from Fluka Chemie (Switzerland) and acetonitrile (water content of 10 ppm) from Burdick & Jackson, Inc. (Muskegon, MI); phosphate-buffered solution (PBS) at pH 7.4, without calcium and without magnesium, from Biofluids, Inc. (Rockville, MD); and gaspermeable teflon capillary tube of 0.8 mm inner diameter
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Fig. 1. An electrochemical cell for the potentiostatic preparation of LiPc using a Pt gauze as the working electrode (W). The working electrodes used were a Pt gauze, Pt coiled-coil, and Pt coil; the details of these electrodes are given in the text. The reference electrode (R) was a Ag1/AgCl(s) and the auxiliary electrode (A) was a Pt wire. The separation between the electrodes X, Y, and Z were about 18, 13, and 13 mm, respectively.
and 0.025 mm wall thickness from Zeus Industrial Products, Inc. (Orangeburg, SC). The electrochemical cell shown in Fig. 1 uses platinum electrodes for both the working and auxiliary electrodes. The auxiliary electrode was a Pt wire, while the working electrode was either a Pt gauze (450 squares/cm2 with a wire thickness of 0.20 mm (32 ga.); 40 mm diameter and 50 mm length), a coiled Pt wire (with a wire size of 0.63 mm (22 ga.); coil i.d. 0.8 mm and length 30 mm), or a helical coiled coil (with a wire size of 0.63 mm (22 ga.); small coil i.d. 0.8 mm, large coil i.d. 10 mm, length 60 mm). No special surface treatment was given to the platinum electrodes; and all chemicals were used as obtained from the manufacturers. Electrochemical synthesis of LiPc LiPc was synthesized by a procedure similar to those reported earlier19,20 with minor modifications: the elec-
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trosynthesis was performed under air and at constant potential resulting in pO2-sensitive crystals. The dilithium salt Li2Pc was oxidized to LiPc in acetonitrile using TEAP as the supporting electrolyte, at a controlled potential using the CV-27 Voltammograph in an electrochemical cell (a 100-ml glass cell vial) using Pt electrodes. Several batches of LiPc were prepared at an oxidation potential that was maintained at a given voltage, from 0.05 to 0.75 V versus Ag1/AgCl(s) electrode. Platinum electrodes were used for both the working and auxiliary electrodes. The auxiliary electrode was a coiled Pt wire inside a fritted glass, while the working electrode was either a Pt gauze, a Pt coil, or a Pt coiled coil. To a 70-ml volume of acetonitrile containing 0.1 M TEAP, 200 mg of Li2Pc were added with constant stirring to obtain a dark blue solution. The stirring continued for about 1 h to completely dissolve the Li2Pc. The cell was kept at room temperature (no special efforts were undertaken to thermostat the vessel) and the electrolysis initiated at constant potential and continued till the completion of the reaction, where the intensity of the electrolysis current was about the same as the residual current. The contents of the cell were not stirred but were covered during the electrochemical oxidation process to avoid dirt and reduce evaporation rate. The completion of the reaction took several days at the lower potential and hours at the higher potentials, 6 –7 days at 0.05 V and about 1 h at 0.75 V. Crystals of LiPc were deposited onto the electrode surface or dropped out of solution as LiPc is not soluble in acetonitrile. As expected, for the same potential, the working electrode with the largest surface area led to the fastest reaction. The LiPc crystals thus obtained were filtered, washed several times with 10-ml portions of acetonitrile, and dried at 85°C overnight. EPR sample preparation Quantitative measurements of the dependence of the EPR line width of LiPc as a function of pO2 were carried out. LiPc samples kept in vials covered with air-tight septa were equilibrated with O2/N2 mixtures of various compositions using hypodermic syringe needles as inlet and outlet. For RF EPR measurements the resonator sizes allowed the use of 15-mm diameter vials and the LiPc samples in the vials were loaded into the resonator directly. For X-band EPR measurements, a suspension of LiPc in PBS (100 ml) was drawn into a gas-permeable, flexible Teflon capillary tube. The tube was folded twice so that the sample occupies the active volume of the cavity. The folded tube was inserted into a 4-mm quartz tube open at both ends and was inserted into the rectangular EPR cavity configured to remain horizontal. The quartz tube containing the sample was slowly purged
with O2/N2 mixture of a desired composition at one end. Equilibration of the system with the available O2 whether at RF or X-band frequencies was ascertained by making sure that the amplitudes reach a constant value. For our experimental set up, this was achieved within a few minutes, but purging with the gas continued for 20 min and throughout the measurement time.
EPR measurements X-band cw EPR measurements were carried out using a Varian E9 spectrometer with TE102 microwave cavity, at a nonsaturating microwave power of 0.2 mW and a field of 3375 G. Due to the inhomogeneity of the magnetic field of the X-band EPR spectrometer over the sample volume, the spectra were deliberately over modulated to broaden out the spectral splittings caused by the inadvertent spatial encoding. The RF (289 MHz) cw EPR measurements were made on a home-built spectrometer, using a 15-mm helical coil resonator with 3.13 kHz field modulation and phase-sensitive detection. The time-domain EPR measurements were obtained using a home built RF (300 MHz) EPR spectrometer, described elsewhere,21,22 that uses a 1-GHz sampler with a channel limitation of 4 k points, corresponding to 4.096 ms. Peak heights reported are peak-to-peak of the first derivative of the absorption mode in the case of both of the RF and X-band cw EPR experiments; the line width in the pulsed RF EPR experiments is the full width at half maximum (FWHM) of the unmodulated absorption line. RESULTS
Several batches of LiPc crystals synthesized at different oxidation potentials were studied for their line widths and oxygen response using three different EPR spectrometers: a cw X-band (9.3 GHz), a cw RF (289 MHz), and a time-domain RF (300 MHz). The response of the line widths of LiPc crystals to oxygen was studied from 0% pO2 (obtained by purging with N2 gas) to 100% pO2 (obtained by purging with O2 gas) condition. A typical response of the line width of an LiPc batch that was found to be pO2-sensitive is shown in Fig. 2. This LiPc batch, synthesized at a constant potential of 0.35 V using a Pt gauze working electrode, was almost exclusively composed of crystals whose line width was sensitive to pO2, the line width ranging from 50 mG to 1.3 G from 0% to 100% O2, respectively. However, an LiPc batch, synthesized at a constant potential of 0.15 V using a coiled-coil working electrode, gave mainly a broad line with a line width of 300 mG which was pO2-independent. In addition, a minor component with a narrow line whose line width and amplitude depend on pO2 was
Lithium phthalocyanine: synthesis and oxymetry
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Fig. 2. Continuous wave (cw) X-band EPR spectra of an oxygen-dependent LiPc batch (electrochemically synthesized at a constant potential of 0.35 using a Pt gauze working electrode) under different pO2 levels. The oxygen percentages from the narrowest to the broadest spectra were: 0.10%, 0.21%, 2.04%, 5.02%, 21%, and 100%, and the corresponding receiver gains were 1.25 3 103, 1.25 3 103, 2.5 3 103, 4 3 103, and 2.5 3 104, respectively. The spectrometer settings were: modulation amplitude, MA, 0.8 G; modulation frequency, MF, 25 kHz; microwave power, P, 0.2 mW; sweep width, SW, 20 G; time constant, TC, 1 s; scan time, ST, 4 min. The spectra were severely overmodulated to avoid multiplets caused due to magnet inhomogeneity, and thus were used only in broad qualitative terms. The inset shows the spectra of an LiPc batch (electrochemically synthesized at a constant potential of 0.15 V using a coiled coil working electrode) that is predominantly composed of oxygen-insensitive crystals of LiPc at 0% O2 and 21% O2 (compressed air); the broad and narrow lines correspond to the oxygen-insensitive and oxygen-sensitive crystals, respectively.
observed. The inset in Fig. 2 shows a typical EPR spectra of an LiPc batch that partly responds to oxygen, under two different pO2s, 0% O2 and air (about 21% O2), respectively. The LiPc crystals, synthesized by electrolysis at 0.35 V that exhibited predominantly pO2-dependent EPR line widths, were examined for their spectral characteristics at different oxygen levels by EPR spectroscopy at RF and X-band frequencies and the results are shown in Fig. 3. The line width of the LiPc crystals were found to linearly increase as a function of pO2. The inverse of the amplitude, which is more sensitive to changes in pO2, also increases with pO2 and is shown in Fig. 3, for both the X-band and RF cw EPR measurements. The homogeneity of the magnet of the X-band EPR spectrometer was not sufficient enough to reliably measure the line widths of the paramagnetic center in LiPc at low pO2 with expected line widths of less than 20 mG. In fact, undesired line splitting was observed due to the spatial encoding of the LiPc particles due to the inhomogeneity of the magnetic field. To estimate the dependence of the spectra on pO2, we deliberately overmodulated the spectral lines with modulation amplitudes higher than the line width to get an envelope of the spectra, and then used only the amplitudes of these broadened lines to correlate to the pO2. The amplitude of the lines correlated inversely to the pO2. However, these
data were not utilized to measure any relaxation times or to evaluate broadening mechanisms. In time-domain EPR methods, saturation effects and modulation artifacts on the spectral line widths are avoided. Therefore, the same batches of LiPc were also studied using a pulsed RF FT EPR spectrometer operat-
Fig. 3. The inverse amplitude (peak-to-peak height) as a function of percent oxygen from the X-band continuous wave (cw) and the RF cw EPR spectrometers (with spectrometer settings: MA 0.5; MF 3.13 kHz; P 2 13 dBm; SW 20 G; TC 640 ms; ST 500 s). The oxygen-sensitive batch shows an amplitude and line width that strongly depend on pO2. The inverse of the peak heights of the first derivative EPR signal versus percent oxygen for both the cw X-band and cw RF EPR cases show a linear increase with pO2.
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Fig. 4. The FT spectra of the oxygen-sensitive LiPc batch shown in Fig. 2, using a time-domain RF EPR spectrometer at two different oxygen percentages, 0% O2 and 2.04% O2, respectively. A 1-GHz sampler with a channel limitation of 4 k points, corresponding to 4.096 ms was used to collect the data. The wiggles in the 0% O2 data are due to truncation of the FID at 4.1 ms, no digital filters were used.
ing at 300 MHz. Similar to the cw EPR measurements, the crystals show pO2-dependent line widths and amplitudes. Typical FT spectra are shown in Fig. 4 at two different pO2s of 0% O2 and 2.04% O2 for the same batch whose cw spectra and its oxygen response are shown in Figs. 2 and 3, respectively. The pO2 dependence of the line width of the FT spectra obtained with the pulsed EPR spectrometer is shown in Fig. 5. The line shape of the spectrum obtained from LiPc crystals at low pO2 using time-domain RF EPR spectrometer were fitted to a single exponential decay constant. The line width of the FT spectra increases with increasing pO2. The line
Fig. 5. A plot of the full width at half maximum in kilohertz versus percent oxygen. The line width under nitrogen atmosphere was 350 kHz and increases linearly, with a sensitivity of 195 kHz (70 mG) per percent oxygen, R 2 5 0.996, as the percent oxygen is increased reaching 2400 kHz at 7.5%. Above 7.5% O2, the signal was still measurable but the spectra obtained were so noisy that determining the line widths from them was not even attempted.
widths of the spectra from the time-domain RF EPR data were plotted versus percent oxygen and fitted to a straight line, which gave a sensitivity of 195 kHz (70 mG) per percent oxygen (R2 5 0.996). As the pO2 was increased, the free induction decay (FID) survives for shorter time intervals (the line width increases), decreasing the amplitude of the detected signal substantially because an appreciable fraction of the signal was lost within the spectrometer dead time. As the pO2 is increased to 7.5% and higher, the signal-to-noise was prohibitively low to do accurate line width measurements. Batches of LiPc whose spectral properties did not show strong pO2 dependence, in which the broad (300 mG) pO2-independent line is the dominant component of the product in the cw measurements, do show pO2 dependence in the pulsed mode. Whereas the cw measurements detect both the broad and narrow lines, the pulsed mode more favorably detects narrow lines because the FIDs corresponding to narrow lines persist beyond the spectrometer dead time, while those corresponding to broad lines decay completely within the dead time. Thus, whereas the cw measurements show the narrow lines as peaks on top of the broad lines, the pulsed EPR measurements show the narrow lines almost exclusively. The dependence of the electrolytic conditions on the yield of the LiPc crystals and their spectral properties was investigated by varying the electrode geometry as well as the electrolysis potential. The variation of the Pt working electrode from a gauze, to a coil, to a coiled coil gave different types of crystals with respect to their response to pO2. The Pt gauze gave pO2-sensitive crystals of LiPc over a broad range of potential. Oxygensensitive LiPc was obtained at all potentials used (0.05– 0.55 V) to varying fraction of the total product. Electrolysis in the range of 0.25– 0.35 V gave crystals that were predominantly pO2-sensitive, whereas the lower potentials gave crystals that were predominantly pO2-insensitive. Electrolysis with the helical coiled coil, on the other hand, gave pO2-sensitive crystals at potentials above 0.25 V; at potentials below 0.25 V the crystals obtained were almost exclusively pO2-insensitive, while those synthesized above 0.35 V were predominantly pO2-sensitive. The crystals obtained using the coiled coil were larger compared with those obtained by using a gauze. Electrolysis with the simple helical coil gave pO2-sensitive crystals at potentials above 0.35 V. Here again, at 0.25 V and below the crystals obtained were exclusively pO2-insensitive. The size of the crystals obtained using the simple coil were similar to those obtained using helical coiled-coil. Attempts to synthesize the LiPc using the same set up as above, but in a constant current mode at 5, 20, and 50 mA for 24, 48, and 72 h, respectively, produced LiPc crystals but failed to produce pO2-sensitive crystals. At-
Lithium phthalocyanine: synthesis and oxymetry
tempts were also made to see whether the pO2-sensitive crystals can be separated from the mixture of products containing both pO2-sensitive and pO2-insensitive batches by recrystallization from a suitable solvent such as chloronaphthalene. But such attempts were not successful in obtaining useful product for oxymetry, because the recrystallized products exhibited EPR spectra whose line widths were not pO2-dependent. DISCUSSION
The electrochemical synthetic route for the preparation of LiPc described in this paper shows the feasibility of obtaining useful quality particulate spin probes for oxymetry in a relatively simple and inexpensive manner. The oxidation of Li2Pc to LiPc has been found to be a straight forward reaction dependent mainly on the surface area of the working electrode and the potential applied. The Pt gauze with the largest surface area leads to shorter oxidation times at a given potential because the LiPc crystals formed are deposited on the surface of the electrode and the Pt gauze has the largest surface area. The crystals formed can be classified into two types based on the response of their EPR line width to pO2. For all three working electrodes used, those batches that were synthesized at lower potentials (0.05– 0.15 V) were predominantly, and in some cases almost exclusively, the pO2-insensitive crystals; batches that were synthesized at intermediate potentials (0.25– 0.35 V) gave mainly the pO2-sensitive crystals, while those synthesized above 0.35 V gave mixtures of the two types, with increasing proportion of the pO2-insensitive crystals as the potential is raised. At 0.75 V, using a coiled coil, the product was mainly pO2-insensitive. Contrary to reports in the literature, which recommend electrolysis at constant current,23 the results from this study indicate that pO2sensitive LiPc can be synthesized potentiostatically. Crystal structure data of the lithium complex of phthalocyanine radicals reported in the literature give two different forms: a tetragonal of space group P4/nnc, with a 5 b 5 19.6 Å, c 5 6.4 Å 19,23 and a tetragonal of space group P4/mcc, with a 5 b 5 13.8 Å, c 5 6.403 Å. 23 The former structure gives a pO2-independent EPR signal (300 mG) while the latter structure gives a pO2dependent EPR signal (20 mG to 1.3 G). Based on the reported space groups, the pO2-sensitive batches we prepared are consistent with the P4/mcc space group, whereas those batches that give pO2-independent EPR signal are consistent with the P4/nnc space group. The line width of the pO2-sensitive LiPc, unlike many other carbon-based paramagnetic materials, and spin probes such as triacetoneaminoxyl (TANO)23 and other nitroxides, increases linearly as a function of pO2 from 0% to 100% pO2. In the absence of oxygen, the line
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width of the pO2-sensitive LiPc was estimated to be approximately 50 mG. This width is broader than the reported line width of 14-20 mG8,16,23 due to the inhomogeneity of the magnetic field of the X-band EPR spectrometer. Because of the inhomogeneity of the Xband magnet used in the current study, attempts were not made to measure line widths as a function of pO2. The EPR spectra were overmodulated and amplitudes were used to study the spectral response to pO2. Other workers24 have presented detailed instrumental considerations at X-band to reliably measure the linewidth of LiPc at very low pO2. All batches of LiPc synthesized by the above potentiostatic method were found to be stable at room temperature for periods of several months, the period they were studied, making them useful probes for oxymetry, an advantage compared to other probes such as fluoranthene hexaflourophosphate which are not quite stable at ambient temperatures. LiPc can also be used as a standard in low frequency pulsed EPR systems because it has a long spin-spin relaxation time constant (T 2 ) relative to the spectrometer dead time; it has an FID that lasts for several microseconds that persist beyond the dead time at such frequencies that are of the order of 500 ns. The line shape of the spectrum obtained from LiPc crystals that are subject to minimum pO2 were fitted to a single exponential decay constant corresponding to a one-dimensional linear Heisenberg magnetic exchange. Any attempt to modify the lattice would destroy the exchange coupling resulting in much broader lines that are probably not sensitive to pO2. From the nature of the results obtained from the EPR and electrolysis experiments, it is possible to draw some broad, albeit useful, conclusions. The grain size of the LiPc crystals was found to depend on the actual geometry/shape of the Pt electrode used, namely gauze, coil, coiled-coil. A pO2-sensitive LiPc was produced for the broad range of working potential from 0.10 to 0.75 V in different proportions. The range of potential used also decided the net yield of pO2-sensitive LiPc. The optimal range of potential that gave maximum percentage yield of pO2-sensitive LiPc was 0.25– 0.35 V using the Pt gauze electrode. At potentials lower than 0.25 V the product was mainly pO2-insensitive; at potentials above 0.35 V the percentage of the pO2-sensitive fraction was reduced. Electrochemical procedures described in this report should be useful to prepare these probes with relative ease and good yield making oxymetric studies widely accessible. Further, the EPR methods described to characterize the products for oxymetric applications will help in choosing appropriate experimental conditions to obtain LiPc crystals with pO2-sensitive paramagnetic centers.
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