J. Phys. Chem. C 2008, 112, 6605-6612
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Improved Production and Separation Processes for Gadolinium Metallofullerenes James W. Raebiger and Robert D. Bolskar* TDA Research, Inc., 12345 West 52nd AVenue, Wheat Ridge, Colorado 80033 ReceiVed: August 10, 2007; In Final Form: February 8, 2008
A comprehensive scheme for processing all arc-produced gadolinium monometallofullerenes into separate quantified fractions is presented. As generated by the carbon arc process, endohedral metallofullerenes are entrained in a complex mixture of more abundant empty fullerenes and carbonaceous soot. The process described herein exploits the differences in solubility and redox reactivity between different classes of Gd@C2n and empty fullerenes to effect their separation from one another. Importantly, the processes not only facilitate use of the normally soluble metallofullerenes, such as Gd@C82, but also provide access to the normally insoluble metallofullerenes, such as Gd@C60. In quantifying the Gd@C2n contents of the different obtained fractions, the normally soluble Gd@C2n are found to be about a 10% minority of the total arc metallofullerene product, while the normally insoluble Gd@C2n comprise up to as much as 90% of the total amount of arcproduced Gd@C2n. Thus, new access to the insoluble class of Gd@C2n in addition to the soluble fullerenes now increases the total availability of monometal Gd endohedrals by up to an order of magnitude.
Introduction Endohedral metallofullerenes are closed carbon fullerene cages that have metal atom(s) entrapped in their interior spaces.1 Among the most prominent characteristics of metallofullerenes is the charge transfer from their interior electropositive metal atoms to their electronegative fullerene cages. Metallofullerenes of the lanthanide elements are of particular interest, because they can be made in relative abundance and have potential applications in biomedicine and materials science. Important examples of biomedical applications under study include Gd metallofullerenes as paramagnetic contrast agents for magnetic resonance imaging (MRI)2-8 and various lanthanide metallofullerenes for nuclear medicine.9,10 For these and other applications, metallofullerenes offer stable entrapment of otherwise toxic metal ions along with the rich exterior functionalization chemistry of the fullerene cages. The carbon arc discharge process is the most commonly employed method to produce macroscopic amounts of metallofullerenes. In a variant of the process by which Kra¨tschmer, Huffman and co-workers11 first demonstrated the macroscopic generation of C60 and other empty fullerenes, metal-doped graphite rods are used in place of pure graphite rods to generate a mixture of empty fullerenes, endohedral metallofullerenes, and carbonaceous soot. Alternatives to the arc discharge process include the radiofrequency furnace method, which is well-suited for the production of metallofullerenes with low melting and boiling metals;12 a plasma method that uses fullerenic soot as the carbon feedstock;13 and the laser vaporization method, with which metallofullerenes were first generated in macroscopic quantities.1c The bulk production methods generate complex mixtures of empty fullerenes and metallofullerenes entrained together in a carbonaceous soot matrix. For development of the biomedical and other applications of metallofullerenes, separating the endohedrals from the empty fullerene cages is necessary, and further separation into fractions with individual endohedral * To whom correspondence should be addressed: e-mail bolskar@ tda.com.
cages and/or isomers is highly desirable. The major separation tool applied to these problems for more than the past decade has been chromatography. Multistage HPLC is particularly effective for isolating M@C82 species, because of their ready solubility in “normal” fullerene solvents such as toluene and other methylated aromatics; accordingly, the majority of attention has focused on the soluble endohedrals.14,15 However, the soluble M@C2n (particularly several isomers of M@C82) comprise only a small minority of the total metallofullerenes generated by the arc process. In previous work, Diener and Alford16 estimated that soluble metallofullerene species, chiefly Gd@C82, comprise only 4% of the total xylene-extractable arcproduced and sublimed Gd metallofullerenes. Thus, a highly significant amount of the Gd metallofullerenessover 90%s have remained unused to this point, when only Gd@C82 is recovered and purified by conventional extractive and chromatographic methods. Since one of the potential obstacles to the study and implementation of metallofullerene materials in applications including medicine is their relatively restricted availability, we now report a new process that gives access to all arc-generated metallofullerenes. With our recent studies of metallofullerene-based MRI contrast agents and their waterproton relaxivity mechanisms, our work to develop improvements to the production and separation process has focused on monometal fullerenes of gadolinium.5 The processes detailed in this work offer several important advantages over prior methods. The new processes do not expose the metallofullerenes to nucleophilic, reactive solvents such as amines, and they do not exclusively depend on chromatography. Most importantly, these methods provide access to the total amount of useable metallofullerenes, thereby increasing access to endohedrals by up to an order of magnitude for the example of Gd metallofullerenes. This effort expands on our previous work with gadolinium monometallofullerenes.17 Experimental Section Materials and Methods. Graphite rods (6.35 mm × 153 mm, 48% porous) were purchased from Toyo Tanso USA. Gadolinium(III) nitrate hexahydrate (99.9% Gd) was purchased from
10.1021/jp076437b CCC: $40.75 © 2008 American Chemical Society Published on Web 04/08/2008
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SCHEME 1: Flowchart for the Separation of Gadolinium Metallofullerenes
Figure 1. Mass spectrum of ODCB-insoluble fullerenes from the extraction of arc sublimate.
Strem Chemicals and used as received. Anhydrous solvents were obtained from Sigma-Aldrich and used as received. Inert atmosphere manipulations were conducted in a Vacuum Atmospheres glovebox under argon (O2, H2O < 1 ppm). Mass spectrometry was performed on a custom-built laser-desorption combination linear and reflectron time-of-flight mass spectrometer. Mass spectra samples were briefly ball-milled in a vial containing alumina beads and ca. 50-fold excess of 7,7,8,8tetracyanoquinodimethane (TCNQ); the resulting mixture (excluding the alumina beads) is pressed onto a stainless-steel target for analysis. HPLC was performed with a Waters 510 HPLC system using a 10 mm × 250 mm Cosmosil Buckyprep column (Nacalai Tesque Inc.) with toluene eluent. UV-vis-near-IR spectra were recorded on a Perkin-Elmer Lambda 19 spectrometer. Elemental analyses were performed at Huffman Laboratories (Golden, CO); samples were digested in acid prior to analysis by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Thermogravimetric analysis (TGA) was conducted with a Shimadzu TGA-50 thermogravimetric analyzer. All E° values are quoted versus the ferrocene/ferrocenium couple ) 0.0 V. Metal Doping of Graphite Rods.10a Graphite rods were placed in a Schlenk tube, which was evacuated for 1 h. A solution of 2 M Gd(NO3)3 in MeOH was added to the evacuated rods. After being soaked for 2 h, the rods were removed and allowed to air-dry. Then they were heated under vacuum (10 mTorr) at 900 °C overnight. Rod gadolinium enrichment levels are calculated as the mass increase due to the incorporation of Gd2O3 into the rods. Electric Arc Discharge Synthesis of Gd@C2n. Gd-fullerenecontaining soot was generated by the standard direct-current (DC) arc discharge method with a custom-built arc apparatus operating at 150 Torr of helium and 175 A. The Gd-impregnated rods were arced with periodic back-burning (reverse-polarity arcing) of cathode deposits to maximize the yield of fullerenes and metallofullerenes.18 Sublimation of the raw arc-produced soot onto an isolated, water-cooled cold finger inside the arc chamber at 750 °C and 5 mTorr separated the fullerenes and metallofullerenes from the non-fullerene carbon soot. Solvent Extraction of Sublimate. Anaerobically collected sublimate was extracted with o-dichlorobenzene (ODCB) inside a glovebox by use of a Soxhlet apparatus, operating at 30 Torr to lower the ODCB boiling point, until the washings were colorless. This removed most of the soluble fullerenes such as C60 and C70, as well as soluble Gd@C82. The soluble fullerenes were isolated as a solid mixture by vacuum-distilling off the solvent. Both the soluble and insoluble solids were rinsed with hexane and dried under vacuum at 200 °C for 2 h to remove residual ODCB.
Figure 2. Mass spectrum of ODCB-soluble fullerenes from the extraction of arc sublimate.
Separation of Enriched Gd@C60 Material. ODCB-insoluble material (0.50 g) from arc sublimate extraction was stirred vigorously in 20 mL of ODCB, to which AlCl3 (30% by weight; 0.15 g) was added. A deep brown solution color formed and the mixture was stirred for several hours, after which it was washed with an additional 30 mL of ODCB in a Soxhlet extractor until the extracts were colorless. The isolated insoluble solids were washed with tetrahydrofuran (THF; to remove any residual AlCl3) and with hexane. The ODCB solution was reduced to dryness by vacuum distillation, and the resulting solid was washed with THF and hexane. Both solids were separately dried under vacuum at 200 °C for 2 h. In an alternative procedure, NOSbF6 was used as the oxidant rather than AlCl3. The NO+ reaction was executed similarly to the AlCl3 reaction, except that 0.20 g of NOSbF6 (40 wt %) was used with 0.50 g of ODCB-insoluble fullerenes. The isolated solids were washed with CH3CN and then hexane, and separately dried under vacuum at 200 °C for 2 h. Isolation of Gd@C82+-Enriched Material. In a typical reaction, 1.00 g of the ODCB-soluble fullerenes obtained from the ODCB extraction of the sublimate was dispersed in 100 mL of CH2Cl2. AgSbF6 (150 mg) was added, and the reaction was stirred vigorously for 2 h. The mixture was filtered, and the filtrate was collected and reduced to dryness under reduced pressure. The resultant solid was washed with 2 × 10 mL of CH3CN, with 4 × 10 mL of toluene (or until the washings were colorless), and finally with 2 × 5 mL of hexane; this yields about 50 mg of dark brown solid, mass spectral analysis of which shows a dominant peak due to Gd@C82. Reduction of Gd@C82+-Enriched Material. In a typical reaction, 50 mg of Gd@C82+-containing enriched material,
Enabling Separation Processes for Gd Metallofullerenes
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TABLE 1: Results of the Isolation of Metallofullerenes for Varying Gd Enrichment Levels of Graphite Rods experiment Gd, mol % mass arced, g sublimate, g yield, % ODCB-insoluble, g (% of sublimate) ODCB-soluble, g (% of sublimate) “Gd@C82+”, mg (% of sublimate) “Gd@C60”, mg (% of sublimate)
A
B
C
D
0.47 67.42 1.80 2.67 0.345 (19.7) 1.34 (74.4) 61 (3.4) 131 (7.3)
0.60 61.13 2.17 3.55 0.612 (28.2) 1.44 (66.2) 64 (3.0) 320 (14.7)
0.70 43.81 1.38 3.14 0.500 (36.4) 0.94 (68.4) 49 (3.5) 213 (15.5)
0.80 56.10 1.57 2.80 0.576 (36.7) 1.00 (63.9) 62 (4.0) 202 (12.9)
predissolved in 50 mL of CH2Cl2, was stirred for 2 h with 25 mg of decamethylferrocene, Fe(C5(CH3)5)2. A precipitate formed, which was isolated by filtration, washed with CH2Cl2 and hexane, and dried under vacuum. Yield ) 35 mg (70%) of black solid that is freely soluble in toluene, which was used as the mobile phase for its subsequent HPLC analysis. The reaction filtrate has the characteristic green color (λmax ) 783 nm) of [Fe(C5(CH3)5)2]+ generated in the redox reaction. Purification of Gd@C82. Reduced, neutral Gd@C82 enriched material (50 mg) was stirred in 50 mL of toluene overnight. It was filtered through a 0.45-µm poly(tetrafluoroethylene) (PTFE) membrane to remove the insoluble material, which amounted to 25 mg. The solution was eluted with HPLC on a semipreparative Buckyprep column with toluene eluent (5 mL/min). The solution was injected into the HPLC (2 mL injection loop), and the eluent fractions between 30.5 and 31.5 min (Gd@C76) and between 33 and 42 min (Gd@C82) were separately collected. The solution was immediately reduced to dryness under vacuum, and the resultant solid was stored under argon prior to collecting the next fraction. This yielded 3 mg of pure Gd@C76 as a black solid and 9 mg of pure Gd@C82 as a brown-black solid. Results and Discussion Arc Production and Separation of Endohedral Metallofullerenes. The electric arc discharge process for producing fullerenes is a well-established method in which graphite rods are resistively heated under a reduced pressure atmosphere of helium to form carbonaceous materials containing up to 20% empty C2n fullerenes.11 When graphite rods pre-impregnated with metal or metal ions are substituted, endohedral metallofullerenes (M@C2n) are produced concurrently with the empty C2n, including C60, C70, and higher C2n (2n g 74).19 One of the biggest issues in metallofullerene science is the separation of metallofullerenes from both the carbonaceous soot products and the empty fullerenes. As is most typically practiced, fullerenes and metallofullerenes are recovered from the mixed soot product by solvent extraction with those solvents commonly known to dissolve fullerenes, such as toluene, haloarenes, and carbon disulfide.20 Detailed studies on the extraction of endohedral fullerenes from arc soot with a variety of solvents and conditions have been reported.21 Metallofullerene extraction efficiencies can be increased beyond those obtained with only arenes or chloroarenes by use of more polar and nucleophilic solvents such as dimethylformamide (DMF),22,23 aniline,21,24 and pyridine,21,24a,25 either alone, in combination with, or successively with other solvents.21,26 Furthermore, metallofullerene extraction efficiency and selectivity can be further increased by employing high-pressure27 and high-temperature conditions,28 as well as ultrasonication29 during soot extraction. However, basic and nucleophilic nitrogen-containing solvents appear to react with electronegative metallofullerenes, including by chemical reduction, reduction followed by proton transfer, and formation of charge-transfer complexes, or likely a combination
of these modes.21,30 The nature of solvent-metallofullerene interaction likely varies with the identity of the endohedral element, fullerene cage (based on both number of carbon atoms and the structural isomer), and solvent. High-boiling and strongly interacting solvents such as those above can be very difficult to completely remove from fullerenes, so our objective was to maximize metallofullerene removal from the crude soot without using these solvents. Rather than directly expose the fullerene-laden soot to organic solvents to separate metallofullerenes, we sublime the empty fullerenes and metallofullerenes out of the soot.1c,31 The mixture of fullerenes and metallofullerenes is collected inside the arc chamber on an internally cooled cold finger that is isolated from soot exposure; a typical mass spectrum of Gd metallofullerenecontaining sublimate is shown in Figure S1, Supporting Information. Harvesting the fullerenes via sublimation avoids their direct contact with solvent while they are in the presence of soot and other byproducts present in the arc soot, particularly the lanthanide carbide. The latter is a concern because, for example, this strongly reducing species was postulated to have generated the highly reactive dichlorophenyl radical during a trichlorobenzene extraction of lanthanum endohedral soot, resulting in formation of small amounts of La@C74 and La@C72 dichlorophenyl adduct derivatives.32,33 A chemical reaction such as this, while quite interesting for the serendipitous synthesis of novel fullerene derivatives, is to be avoided in the overall scheme to separate endohedrals into different classes. Additionally, as a practical matter, it is easier to handle sublimed fullerene powders than bulk low-density soot. Mass spectral analysis shows that only negligible amounts of fullerenes remain behind in the sublimation-treated soot, indicating that sublimation is highly effective at removing fullerenic species from the soot matrix. Discrimination of Fullerenes into Branches. The major focus of this study is post-production processing and purification of metallofullerenes, to ensure that all possible arc-generated materials can be utilized. The separation processes described here are based on solubility differences between fullerene classes, both before and after chemical manipulation of the metallofullerene redox state.17 The flowchart in Scheme 1 illustrates how all of the sublimable empty fullerenes and Gd@C2n metallofullerenes were manipulated into distinct classifications. The sublimate is first portioned into those fullerenes insoluble (branch 1) and those soluble (branch 2) in normal fullerene solvents. Then chemical oxidation (removal of electron(s) to form a cation) is used to convert otherwise insoluble species into soluble and manipulable species, with the solubility changes exploited to effect physical separation. Prior to conducting chemical redox-based separation of the fullerenes, the first step with the sublimate is an exhaustive solvent extraction with o-dichlorobenzene (ODCB), the most potent routinely used fullerene solvent.20 This process separates soluble empty fullerenes (C60, etc.), soluble Gd@C82, and traces
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Figure 3. Mass spectrum of Gd@C2n+ (2n g 72) fullerenes isolated from branch 1.
Figure 4. Mass spectrum of Gd@C60-enriched fullerenes isolated from AlCl3 treatment of branch 1.
of other soluble Gd@C2n from the fullerenes such as Gd@C60 and C74 that are insoluble. Typical yields of soluble and insoluble fullerenes after extraction are listed in Table 1, and the mass spectra of these materials are shown in Figures 1 and 2. Thermogravimetric analysis (TGA) of the solids isolated after solvent extraction demonstrated that they contain significant amounts of ODCB (up to 15% by weight; Figure S2, Supporting Information). Entrapped fullerene solvents can be difficult to remove with vacuum drying at room temperature, so we performed vacuum drying of the solids at 200 °C to a constant mass (requiring about 2 h). Subsequent TGA analysis showed less than 2% additional weight loss (Figure S3, Supporting Information), indicating that the majority of the entrapped ODCB solvent was now effectively removed. This allows for more accurate determination of fullerene masses isolated in each step, because without minimization of the amount of entrapped solvent, the measured carbon content of the material would be elevated and this would dilute the measured Gd content. Gd elemental analysis on the soluble fullerene and metallofullerene fraction, in comparison to the elemental analysis of the raw sublimate, indicated that 10% of Gd metallofullerenes are soluble. These results differ somewhat from the earlier estimate of 4% for soluble Gd metallofullerene content.16 This difference is due to several factors, including the present improved extraction technique using ODCB Soxhlet extraction vs a less exhaustive xylene extraction of the sublimate, as well as minor variation in metallofullerene arc yields that vary with slight differences rod doping and the arc operation. Branch 1 Insolubles: Redox Chemistry for Separation of Metallofullerene Classes. At this point in the processing, the ODCB-insoluble fullerenes, which contain up to 90% of all Gd metallofullerenes, are made up of two different classes, including a minority class that can be oxidized to soluble cations with moderate chemical oxidants. As the majority of Gd metallofullerenes cannot be easily oxidized, removing the fullerenes that are readily oxidizable is facile. We found that oxidants such as silver(I) and tris(4-bromophenyl)aminium (E° ) 0.65 and 0.70 V, respectively, both in CH2Cl2)34 readily oxidize a portion of the insolubles to cations soluble in solvents like CH2Cl2. This soluble cation fraction, (see Scheme 1) contains Gd@C2n metallofullerenes with 2n g 72, the mass spectrum of which is shown in Figure 3. This fraction can be isolated by precipitation upon hexane addition and can optionally be reduced to neutral species with mild reducing agents. However, the yield of these fullerenes is low; only 3-5% by mass (relative to the mass of ODCB-insoluble fullerenes that was treated with oxidant) is isolated by use of these oxidants. This may reflect an actual lower content of fullerenes and Gd metallofullerenes that can
be oxidized by these reagents or the fact that some of these higher metallofullerenes (Gd@C2n with 2n g 72) may be trapped in the insoluble matrix, preventing removal in this step. Nonetheless, the method removes these chemically distinct metallofullerenes from the greater amount of insoluble Gd@C60dominated material, which will facilitate their further separation to individual fullerenes or isolation as derivatives that may have novel cage structures.32,35 Previously, in some cases, M@C60 and M@C70 species have been solubilized directly from arc soot into solvents like pyridine and aniline (but without quantification of the extraction efficiency),24,25 and in one study Eu@C60 was extracted from arc sublimate and purified by HPLC with aniline as the solvent.36 Furthermore, reduction of M@C60 and M@C70 to soluble species by these solvents is supported by evidence for the dissolution of M@C60 and M@C70 as anions.16,37 However, as discussed above, reactive extraction of metallofullerenes with amine solvents was avoided in this work, and instead we developed a chemical means to deplete the other species and leave behind a relatively pure Gd@C60 fraction while avoiding direct chemical transformation of Gd@C60 and related metallofullerenes. After the above solvent washings and oxidant treatments, various empty fullerenes still remain in the insoluble material. At this point, the insolubles contain what we denote the “Gd@C60 class,” which includes Gd@C60, Gd@C70, and Gd@C74 metallofullerenes, as well as the empty small-band gap fullerene C74 and small amounts of empty, normally soluble fullerenes like C60. For applications development with metallofullerenes, we strive to get out as much of the empty fullerenes as possible. Solvent washing alone does not completely remove all potentially soluble empty fullerenes, because some are apparently either entrained physically in the solid mixture or are possibly bonded to the other fullerenes to form insoluble dimers or oligomers. C74 is insoluble in all of the normal fullerene solvents, making it particularly difficult to remove.16 A typical mass spectrum of the ODCB-insoluble fullerenes is shown in Figure 1, which shows that at this stage Gd@C60 and C74 are present at approximately the same abundance. To minimize empty C74 content and further enrich metallofullerene content, we treat this material with a strong oxidant in ODCB in a process that removes a considerable portion (up to 50%) of the C74 and the higher Gd@C2n and leaves insoluble a fraction highly enriched in Gd@C60 as well as Gd@C70 and Gd@C74. As shown above, silver(I) and tris(4-bromophenyl)aminium do not efficiently solubilize a significant proportion of the ODCBinsoluble C2n and Gd@C2n, so reagents with stronger oxidizing power were used.
Enabling Separation Processes for Gd Metallofullerenes
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Figure 5. Mass spectrum of fullerenes solubilized by AlCl3 treatment of ODCB-insoluble material.
Figure 6. Mass spectrum of Gd@C60 enriched fullerenes isolated from nitrosonium treatment of branch 1.
The reaction of the ODCB-insoluble fullerenes with the strong oxidant AlCl3 (E° ≈ 1.1 V in CH2Cl2)38 in ODCB quickly generates a dark brown solution indicative of soluble fullerenes. Subsequent exhaustive solvent extraction and isolation results in the removal of about 30 wt % of the fullerenes. After the oxidative AlCl3 treatment, the mass spectrum of the remaining insolubles (Figure 4) reveals that the ratio of Gd@C60 to C74 is much greater than 1 and that Gd@C60 is the most prevalent species. The fullerenes removed in this step are C60, C70, C74, and Gd@C2n (2n g 72) (see Figure 5). Thus, the treatment of insoluble fullerenes with AlCl3 results in a mixture that is enriched with Gd@C60. The reaction products solubilized by AlCl3 treatment will require further characterization to establish their nature, but the reaction likely proceeds with the oxidation of the fullerene by AlCl3 with in situ formation of tetrachloroaluminate (AlCl4-) as the counteranion.34 In this step, it is important to avoid arenes like benzene or toluene and traces of water to prevent the AlCl3catalyzed polyaddition of arenes to the fullerene cages.39,40 Because it is deactivated for addition chemistry, no reactions between C60 and ODCB in the presence of AlCl3 have been observed.40 While the oxidation power of AlCl3 is well established, its nature as an oxidant is complex and an alternative mechanism cannot be ruled out, such as the incipient generation of a carbocation that functions as the oxidizing agent in the reaction.34 We also used a different oxidant, nitrosonium (NO+), to empirically compare the effects of a well-established oneelectron oxidant to the action of AlCl3 on the insoluble fullerenes and metallofullerenes. The oxidation potential of NO+ (E° ) 1.00 V)41 in CH2Cl2 is similar to that of AlCl3, so if the latter functions solely as an oxidant (as opposed to having a separate chemical reactivity, such as derivatization, causing fullerenes to become soluble), then similar results should be obtained with nitrosonium. When the ODCB-insoluble fullerene mixture is treated with 40 wt % NOSbF6 in ODCB, a dark brown solution color quickly develops, similar to the effect of AlCl3. After workup and mass spectral analysis, the result is nearly indistinguishable from the AlCl3 treatment: the insoluble material is depleted of C74 and higher Gd@C2n (compare Figure 6 to Figure 4), confirming that these reagents act with oxidation to solubilize and liberate fullerenes for removal. Just as with AlCl3, the fullerenes that are removed by this process are C60, C70, C74, and Gd@C2n, 2n g 72 (Figure S4, Supporting Information), and about 40 wt % is removed. Nitrosonium is a useful alternative to AlCl3 because the easily removable byproduct of the oxidation is nitric oxide gas.
Interestingly, both AlCl3 and NO+ appear to have insufficient oxidizing strength to oxidize and solubilize Gd@C60, Gd@C70, and Gd@C74, putting a lower limit on their oxidation potentials to higher than ca. 1 V (note that the first oxidation potential of C60 is +1.26 V in 1,1′,2,2′-tetrachloroethane).42 There is a relatively large decrease in first oxidation potential between empty fullerenes and their M@C2n lanthanide endohedral counterparts (cf. C2-C82, E° ) 0.72 V; Gd@C82, E° ) 0.09 V),43 consistent with formal reduction of the fullerene cages by the metal ion. Despite the lower ionization potentials of metallofullerenes in general and their open-shell radical natures, some are resistant to oxidation in the condensed phase, while others are not. As the normally insoluble C74 and Gd@C2n (2n g 72) are solubilized by reaction with AlCl3 and NO+, they are evidently more susceptible to oxidation than Gd@C60, Gd@C70, and Gd@C74. These oxidants are not strong enough to oxidize C60 and C70,44 but as the other fullerenes are oxidized, these normally soluble empty fullerenes are liberated from the matrix of insoluble fullerenes. This beneficially reduces the content of C60 and C70 contaminants in the Gd@C60-enriched material (see Figures 4 and 6). Branch 2: Soluble Fullerenes and Gd@C82. The initial ODCB washings of the sublimate contain a mixture of empty fullerenes (predominantly C60, C70, and other soluble higher C2n), Gd@C82, and very small amounts of other soluble Gd@C2n (2n ) 72, 76, etc.). While present in smaller amounts than the other metallofullerenes recovered from arc products, the ODCBsoluble Gd@C2n species are also sought for recovery. Chromatographic separation methods, particularly HPLC, excel for the isolation of individual metallofullerene species that are soluble in normal fullerene solvents such as toluene. Preparativescale chromatography can be performed directly on the total mixture of solubles to separate the minor-component Gd@C82, but alternatives that avoid the large efforts in time and materials this requires are desirable. A variety of innovative processes for the enrichment or separation of soluble M@C82 from empty fullerenes have been described, including reduction-based processes37,45 and selective complexation methods.46 The separative power afforded by these processes is based on property differences between soluble M@C82 and soluble C2n, including redox potentials, polarizability, and solubility. We previously described the use of oxidants to selectively remove the minor quantities of Gd@C82 and other easily oxidized soluble metallofullerenes from mixed soluble fullerenes.17 Gd@C82 is separable from the other soluble fullerenes because of the relatively low potential of its first oxidation (+0.09 V) in comparison to the other soluble fullerenes, particularly C60, for which it is over 1 V more difficult to remove an electron
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Figure 7. Mass spectrum of Gd@C82+ enriched fullerenes isolated from branch 2.
from in solution.42,44 Thus, the application of an oxidant of appropriate potential causes selective oxidation of Gd@C82 to Gd@C82+, which under the proper conditions allows facile recovery of the cation salt (see Scheme 1). Ag+ is effective in oxidizing Gd@C82 to the cationic state, which is soluble in CH2Cl2. Small amounts of neutral empty fullerenes are also extracted into CH2Cl2 (as expected, given their moderate solubilities in CH2Cl2),20 so a toluene wash of the oxidized material was used to remove most of these empty fullerenes. It is also likely that small amounts of higher fullerenes are also oxidized to cations by Ag+, as many C2n with 2n g 76 have first oxidation potentials near ca. 0.7 V.47 The result is about 5% yield by weight of material (relative to the source portion of already ODCB-soluble mixed material) that contains Gd@C82 as the dominant fullerenic species, the mass spectrum of which is shown in Figure 7. The electronic absorption spectrum (Figure S5, Supporting Information) shows a peak in the near-infrared at 1270 nm characteristic of Gd@C82+.48 This material is isolated as the SbF6- salt, with infrared spectroscopy of the product showing the characteristic absorption at 655 cm-1 from this anion (see Figure S6, Supporting Information). Obtaining the material that is highly enriched in Gd@C82 saves significant resources that would otherwise be expended in a multistage HPLC purification performed directly on the total soluble mixture of C2n and Gd@C2n, which requires several continuous days of effort, many liters of elution solvent, and finally time-consuming solvent removal (of high-boiling solvents) from large-volume dilute solutions. As an example, a report by Stevenson et al.49 on the automated HPLC separation of M@C2n stated that “∼1-2 mg of the Sc@C2n fraction can be isolated from ∼400 mg of the soluble fullerene and metallofullerene extract in 2-4 days.” For analysis and purification, the oxidized Gd@C82-enriched material was reduced back to the neutral state by use of decamethylferrocene as the reductant. Next, when 50 mg of pre-enriched neutral Gd@C82 was stirred in toluene, half the mass was found to be insoluble. This was unexpected, as it originated from the branch 2 soluble fullerenes. It is possible that insoluble material results from a slight over- or underreduction during conversion back to the neutral species during the process; no attempt was made to resolubilize the insoluble fraction. Mass spectral analysis of the soluble neutral material (similar to the spectrum in Figure 7) shows it to contain ca. 35% Gd@C82, 10% Gd@C76, and 5% empty C2n, with the remaining ca. 50% comprising other Gd@C2n species, based on rough estimates of peak areas. However, HPLC analysis shows that not all of the Gd@C2n passes through the semipreparative Buckyprep column. Figure 8 shows a typical HPLC trace of
Raebiger and Bolskar
Figure 8. HPLC trace of Gd@C82-enriched fullerenes (Buckyprep, toluene, 5 mL/min).
the soluble neutral material; in this particular sample, peak area integration indicates >85% Gd@C82 relative to the other observed fullerenes. There are two partially overlapping peaks from 33 to 41 min, corresponding to two isomers of Gd@C82. A sample from the short peak immediately preceding the Gd@C82 peaks, at 31 min, was identified as Gd@C76 by mass spectrometry; the peak at 45 min was not identified but was presumed to be a higher Gd fullerene. HPLC indicates the sample contains ca. 5% empty fullerenes, in accord with the mass spectrum. When a 25 mg sample of soluble enriched Gd@C82 materials was processed by HPLC, 9 mg of Gd@C82 (Figure S7, Supporting Information) was isolated as well as 3 mg of Gd@C76 (Figure S8, Supporting Information). These isolated yields are consistent with the mass spectral analysis, with about half of the solubles, other Gd@C2n species, not passing through the column. The reasons why the latter irreversibly adhere to the column are not clear, but given the known air sensitivity of one or more isomers of Gd@C82,14,15 it is very likely that these other Gd@C2n are also air-sensitive and their exposure to air prior to HPLC rendered them unable to pass through the column because of oxidation. For example, Gd@C80 was recently found to be very unstable in air.50 There is a relative lack of HPLC-isolated soluble Gd@C2n species other than Gd@C82, and these merit further investigation. The modest yield of pure Gd@C82 emphasizes how little of the soluble Gd@C2n is practically recoverable in the arc product and further emphasizes the importance of the much greater amount of metallofullerene material present in the insoluble Gd@C60 class. Elemental Analysis of Isolated Fullerene Mixtures. The amount of Gd metallofullerenes present in the isolated materials of our two-branch process was determined by gadolinium elemental analysis on the samples. The analyses showed that about 17 wt % of the arc sublimate is Gd metallofullerenes. In a typical preparation, 65% of the material is isolated as soluble fullerenes and 35% as insoluble fullerenes. Our elemental analyses of the branch products showed that the metallofullerene content of the isolated solids is very batch-dependent. ODCBsoluble fullerenes ranged from 1.5% to 11% metallofullerenes, and the ODCB-insoluble fullerenes ranged from 20% to 50% metallofullerenes. Thus, the ratio of the mass of metallofullerenes in the insoluble material to the mass in the soluble material ranges from 2.5:1 to 10:1. This confirms that the majority of the metallofullerenes is present in the insoluble fraction. An important aspect of increasing metallofullerene availability is to maximize the yields of the raw fullerenic products of arc
Enabling Separation Processes for Gd Metallofullerenes discharge. It is well-known that doping the graphite rods with metals suppresses total fullerene yields relative to empty-only fullerene yields obtained with pure graphite rods, so it is very important to adjust the metal doping level to maximize metallofullerene production. We systematically varied the Gd doping level of the graphite rods and determined the yields of the different branches of fullerene classes using our separation processes. Table 1 summarizes the results obtained for graphite rods doped between 0.45% and 0.80% Gd. As can be seen, the Gd metallofullerene contents increased with increasing Gd doping, and the best results were obtained for a doping level of 0.70% Gd (experiment C, Table 1). Also evident is that increasing Gd doping results in increased amounts of total insoluble fullerenes, which results in more Gd@C60-enriched material without seriously impacting the amount of isolated Gd@C82+-enriched material. Due to the nature of the arc process, there is variability in the results from batch to batch. Different results can be obtained for different doping levels and different extraction/reaction techniques, and certainly different results will be obtained for lanthanides other than Gd. Furthermore, the arc apparatus and its operation are dependent on multiple empirical factors, and different overall yields can result from slight differences in apparatus construction and applied arc parameters.51 Significance and Utility of the Method. These improved processes facilitate utilization of all metallofullerenes produced by the arc process for the first time. Before this work, only as few as 10% of all Gd metallofullerenes made by the arc method, the minority phase of soluble M@C2n that are chiefly M@C82 isomers, were routinely accessed. Our methods now provide access to both the soluble minority Gd metallofullerenes in addition to the majority insolublessa factor of as much as 10 times improvement. The processes described in this work should be adaptable to the types of arc-produced metallofullerenes that are different from the monometallofullerenes, including dimetallofullerene species,19b,52 metal-carbide endohedrals,53 and trimetallonitride (“TNT”) endohedral fullerene species.54 For example, Echegoyen and co-workers55 recently demonstrated an electrochemical oxidation-based differentiation of the D5h and Ih isomers of Sc3N@C80, and it may be possible to exploit redox differences56 between different-sized TNT cages to separate them from one another and from empty fullerenes. Furthermore, redox-based processes can also be combined with solubility and chemical reactivity-based processes that are being developed for TNT purification, as the TNTs are soluble in normal fullerene solvents.57 The processes may also serve as means to identify and isolate novel metallofullerene structures, as has been done with derivatization.32,33,35 We also expect the method to extend to separating metallofullerenes of lanthanides of interest besides Gd, for example Y, La, and Ho. The redox separation processes increase the overall availability of the relatively hard-to-obtain monometallofullerenes to include the majority species that are otherwise inaccessible by solvent extraction alone. This has proved valuable in recent studies of their biomedical applications, particularly the paramagnetic MRI contrast-enhancing agents based on Gd@C2n. After application of the above processes involving solvent extraction and oxidation, the resultant Gd@C60-enriched fullerene mixture is amenable to chemical derivatization, as has been done in separate studies of water-soluble Gd metallofullerene derivatives.5 In combination with separate improvements to the arc reactor production capacity (i.e., increasing the throughput and/ or intrinsic yield of metallofullerene products), these processes
J. Phys. Chem. C, Vol. 112, No. 17, 2008 6611 will help enable the further development of metallofullerene science and applications development. Conclusion Solvent extraction of metallofullerenes followed with chromatography, though a powerful means to purify certain M@C82 species, fails to provide access to as much as 90% of the Gd endohedral metallofullerenes generated by the arc process. With this majority of metallofullerenes remaining insoluble in normal fullerene solvents, as typified by Gd@C60, different means to separate them are required. This work details a protocol for accessing all arc-produced metallofullerenes through a combination of sublimation of the fullerenes out of the arc soot, solvent extraction of minority endohedrals from the sublimate, and purification steps using redox chemistry. As demonstrated quantitatively for Gd endohedral metallofullerenes, these processes separate the metallofullerenes into different chemical classes and minimize contamination by empty fullerenes. This increase in access to arc-generated metallofullerenes by as much as a factor of 10 as compared to traditional methods that access only the minority M@C82 species will contribute to the onward development of the imaging agent and other potential medical applications of metal-containing endohedral metallofullerenes. Further refinement of the metallofullerenes into discrete fractions, characterization of individual metallofullerenes, and quantitative processing of other metallofullerenes beyond those of Gd are all targets for further study. Acknowledgment. We thank the National Institutes of Health (Grant 1R43EB005857-01) for support of this work. Supporting Information Available: Additional mass spectra, thermal gravimetric analyses, and infrared and UV/visible/ near-IR spectra of Gd@C82+. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Heath, J. R.; O’Brien, S. C.; Zhang, Q.; Liu, Y.; Curl, R. F.; Tittel, F. K.; Smalley, R. E. J. Am. Chem. Soc. 1985, 107, 7779-7780. (b) Weiss, F. D.; Elkind, J. L.; O’Brien, S. C.; Curl, R. F.; Smalley, R. E. J. Am. Chem. Soc. 1988, 110, 4464-4465. (c) Chai, Y.; Guo, T.; Jin, C.; Haufler, R. E.; Chibante, L. P. F.; Fure, J.; Wang, L.; Alford, J. M.; Smalley, R. E. J. Phys. Chem. 1991, 95, 7564-7568. (2) Zhang, S.; Sun, D.; Li, X.; Pei, F.; Liu, S. Fullerene Sci. Technol. 1997, 5, 1635-1643. (3) Wilson, L. J. Electrochem. Soc. Interface 1999, 8 (4), 24-28. (4) (a) Mikawa, M.; Kato, H.; Okumura, M.; Narazaki, M.; Kanazawa, Y.; Miwa, N.; Shinohara, H. Bioconjugate Chem. 2001, 12, 510-514. (b) Okumura, M.; Mikawa, M.; Yokawa, T.; Kanazawa, Y.; Kato, H.; Shinohara, H. Acad. Radiol. 2002, 9, S495-497. (c) Kato, H.; Kanazawa, Y.; Okumura, M.; Taninaka, A.; Yokawa, T.; Shinohara, H. J. Am. Chem. Soc. 2003, 125, 4391-4397. (5) (a) Bolskar, R. D.; Benedetto, A. F.; Husebo, L. O.; Price, R. E.; Jackson, E. F.; Wallace, S.; Wilson, L. J.; Alford, J. M. J. Am. Chem. Soc. 2003, 125, 5471-5478. (b) Sitharaman, B.; Bolskar, R. D.; Rusakova, I.; Wilson, L. J. Nano Lett. 2004, 4, 2373-2378. (c) To´th, EÄ .; Bolskar, R. D.; Borel, A.; Gonza´lez, G.; Helm, L.; Merbach, A. E.; Sitharaman, B.; Wilson, L. J. J. Am. Chem. Soc. 2005, 127, 799-805. (d) Laus, S.; Sitharaman, B.; To´th, EÄ .; Bolskar, R. D.; Helm, L.; Asokan, S.; Wong, M. S.; Wilson, L. J.; Merbach, A. E. J. Am. Chem. Soc. 2005, 127, 9368-9369. (e) Laus, S.; Sitharaman, B.; To´th, EÄ .; Bolskar, R. D.; Helm, L.; Wilson, L. J.; Merbach, A. E. J. Phys. Chem. C 2007, 111, 5633-5639. (f) Sitharaman, B.; Tran, L. A.; Pham, Q. P.; Bolskar, R. D.; Muthupillai, R.; Flamm, S. D.; Mikos, A. G.; Wilson, L. J. Contrast Media Mol. Imaging 2007, 2, 139-146. (6) Fatouros, P. P.; Corwin, F. D.; Chen, Z. J.; Broaddus, W. C.; Tatum, J. L.; Kettenmann, B.; Ge, Z.; Gibson, H. W.; Russ, J. L.; Leonard, A. P.; Duchamp, J. C.; Dorn, H. C. Radiology 2006, 240, 756-764. (7) Anderson, S. A.; Lee, K. K.; Frank, J. A. InVest. Radiol. 2006, 41, 332-338. (8) Shu, C.-Y.; Zhang, E.-Y.; Xiang, J.-F.; Zhu, C.-F.; Wang, C.-R.; Pei, X.-L.; Han, H.-B. J. Phys. Chem. B 2006, 110, 15597-15601.
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