Not only are they distinctive and fun to look at, but their PAH-like behavior can also be used to characterize chromatography stationary phases.
Yoshihiro Saito Hatsuichi Ohta Kiyokatsu Jinno TOYOHASHI UNIVERSITY
OF TECHNOLOGY
(JAPAN)
I
n 1985, Kroto and co-workers first observed C60 as a stable ion in the mass spectrum of laser-vaporized graphite, although in 1970 Osawa predicted its existence as a cluster molecule with a round, hollow shape (1, 2). Since Krätschmer and co-workers established a reproducible preparation method in 1990, the molecule has attracted the interest of a huge number of scientists in a wide variety of fields (3, 4 ). Because the cluster molecule contains carbon atoms arranged to form 12 isolated pentagons and 20 hexagons, it was named “Buckminsterfullerene”, “fullerene”, and “Bucky ball”, honoring Richard Buckminster Fuller, a pioneer in the design of geodesic domes. At almost the same time, Krätschmer and co-workers also found C70, the second-most abundant molecule formed by resistive heating of graphite, followed by the discovery of other new members of the fullerene family, such as C76, C78, C82, C84, and their isomers (3, 4 ). Separating fullerenes to obtain the individual members is a critical step to characterize them for applications. HPLC has been widely used to separate them. In addition, many novel stationary phases have been designed and synthesized to effectively separate and isolate fullerenes on the basis of molecular shape (5, 6). On the other hand, an attractive feature of fullerenes is that they can be used as sample probes to characterize HPLC stationary phases, because as a group, they act like polycyclic aromatic hydrocarbons (PAHs) with specific features
© 2004 AMERICAN CHEMICAL SOCIETY
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Retention time (min) FIGURE 1. Typical chromatograms for the separation of fullerene mixtures on (a) monomeric and (b) polymeric ODS columns. Mobile phase, toluene/acetonitrile (45/55); flow rate, 1 mL/min; UV detection, 325 nm; room temperature. (Adapted from Ref. 12.)
and dimensions. For example, fullerenes were used as the analyte in the conformational analysis of the conventional octadecylsilane (ODS) bonded phase at various temperatures, and the stationary phase’s unique selectivity was confirmed. In this article, we will review recent advances in separating fullerenes, retention behavior studies, and the use of fullerenes as sample probes and bonded-phase ligands.
Separating fullerenes The first attempt to separate the most abundant fullerenes, C60 and C70, from Soxhlet extracts of carbon soot was performed with classical open-column chromatography by using alumina or silica as the stationary phase, primarily because the procedure is a convenient laboratory-scale isolation method (7, 8). More effective separation and isolation methods were demonstrated with chemically bonded stationary phases in HPLC. Hawkins and co-workers reported a phenylglycine-derivatized bonded phase for separating C60 and C70 (9). Cox and co-workers used 268 A
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dinitroanilinopropyl silica as the stationary phase (10). Although many types of conventional ODS (11, 12) and polymer-based phases (13, 14) were also used, several phases especially designed for fullerene separation have been developed on the basis of systematic retention behavior studies (15–24). Taking advantage of the electronic interaction between the bonded-phase ligand and the fullerene molecule, Welch and Pirkle synthesized a novel tripodal π-acidic stationary phase, the so-called Buckyclutcher, for preparative separation (15 ). This phase offers simultaneous multipoint interaction with fullerenes for increased retention and separation power. Kimata and coworkers specially designed a high-capacity 3-[(pentabromobenzyl)oxy]propylsilyl (PBB) derivatized silica stationary phase for the preparative separation of fullerenes (16, 17 ). PBB was synthesized on the basis of a systematic survey of fullerene solubility in various organic solvents that contained a heavy heteroatom, such as bromine. The fullerenes’ unique electron-donating and -accepting characteristics have enabled other aromatic stationary phases to be synthesized. Efficient retention was obtained because of the preferable π–π interaction between the large aromatic bonded phase, containing two or more aromatic rings, and the large aromatic solute molecules, fullerenes. One typical phase is the 3-(1-pyrenyl)propyl bonded phase developed by Tanaka and co-workers (17 ). These large, aromatic, commercially available bonded stationary phases are an important tool in fullerenerelated research (15–19), although the conventional open-column technique is still popular, especially for crude separations. In general, separating fullerene isomers becomes more challenging as the molecular weight increases, because the number of isomers theoretically expected dramatically increases. Furthermore, the shape difference in those isomeric species becomes more “unclear” as the molecules get larger. Multistep separations such as 2-D and recycling techniques have been widely applied to separate a single isomer from the native complex mixture; however, the entire, complete separation is still a challenge.
Structural analysis of bonded phases Because of their specific molecular size and the availability of many homologous structures, fullerenes can act as sample probes to characterize novel stationary phases, much the same way that PAHs are used (20–24). When C60, C70, and higher fullerenes were separated on conventional ODS, monomeric ODS with a higher carbon content (i.e., higher surface coverage) exhibited good separation power for C60 and C70 and limited separation for the isomers of higher fullerenes (11, 12). Most polymeric phases exhibited limited retention values for these solutes, although some exhibited better shape selectivity for fullerene isomers. Typical chromatograms clearly show the general trend—on the monomeric phase, fullerenes elute according to their molecular weights; on polymeric phases, elution is based on molecular shape recognition (planarity) (Figure 1). These trends are in good agreement with the general retention behavior for typical PAHs at normal operating temperatures. Monomeric phases separate PAHs mainly by molecular
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FIGURE 2. van’t Hoff plots for C60 (red diamonds), C70 (red triangles), and three PAHs (blue) on a monomeric ODS phase with 2 20% carbon content and surface coverage of 2.8 µmol/m . (Adapted with permission from Ref. 31.)
size, and polymeric phases can distinguish relatively small shape differences, although bulky analytes may exhibit limited retention (25 ). Monomeric phases are synthesized from monofunctional silanes under nonaqueous conditions, and polymeric phases are synthesized under aqueous conditions with trifunctional silanes as the starting material. For polymeric phases, any water in the system then initiates the partial polymerization of the silanization reagent (normally trichlorooctadecylsilane). Because the silane molecule is trifunctional, one or more silanols could be formed in the presence of water, partially polymerized and/ or cross-linked to create octadecyl ligands, forming a rigid phase structure on the silica gel support. In monomeric synthesis, however, the ligands are linked to the support surface only by a single siloxane bond (5, 25 ). The results from chromatographic studies are in good agreement with the interpretation of the phase structure differences based on the synthetic processes. The results also suggest the validity of the systematic analysis of the retention behavior studies of PAHs and fullerenes for characterizing ODS phases, although many other parameters for describing the nature of the solutes and bonded phases should be further considered. Because the molecular size of the fullerenes is quite comparable to the space between neighboring C18 ligands on the silica support, it can be assumed that the surface coverage of the bonded ligands could play an important role in adjusting the space, which may distinguish the size of fullerenes. Introducing phenyl groups at the bottom of the bonded-phase ligand, Saito and co-workers prepared octadecyldiphenyl (C18 diph)-, octyldiphenyl (C8 diph)-, and butyldiphenyl (C4 diph)-silica phases
and compared their retention behaviors with those observed on octadecyldimethyl (C18)-, octyldimethyl (C8)-, and butyldimethyl (C4)-silica phases synthesized with the same silica gel support (26, 27 ). The results clearly indicate that two phenyl rings bonded to the silicon atom induce the spacing uniformity of the alkyl chains, giving good retention for fullerenes because the phenyl rings act to space the bonded-phase ligands on the silica support. The results also suggest the contribution of the alkyl ligand length on retention. A similar trend is confirmed with ODS phases with different surface coverage values (28, 29). Researchers used fullerenes as sample probes and found that a cavity-like structure of bonded ligands of a size comparable to fullerenes was formed (21). For example, the energy-minimized conformation of the bonded phase and its interactions with the fullerenes could explain the good molecular shape-recognition capability of the dimethoxylated phenylpropyl bonded phase (DMPP; 22).
Phase structure at different temperatures In general, analyte retention increases as the column temperature decreases. However, some exceptions in which fullerenes were used as sample probes have been reported. Pirkle and Welch reported an abnormal retention change with decreasing column temperature when they separated C60 and C70 (30). Jinno and co-workers also observed an exceptionally small change in the retention of these fullerenes on DMPP (23) and a multi-legged biphenyl phase (20) at different column temperatures. Ohta and co-workers studied the retention behavior of fullerenes from –70 to 80 °C and confirmed a unique
Because of their specific molecular size and the availability of many homologous structures, fullerenes can act as sample probes to characterize novel stationary phases. relationship between temperature and retention on monomeric ODS phases (26–29, 31). They found the existence of a critical temperature at which the retention of each fullerene molecule was maximized (Figure 2); however, this maximum retention temperature differed, depending on the surface coverage of the ODS phase. Because linear van’t Hoff plots were obtained for small PAHs, the fullerenes’ abnormal behavior suggests that the bulky fullerene molecules are excluded only at lower temperatures and that the ordered bonded-phase structure of the ODS phase is present in the temperature range. Actually, on a typical monomeric ODS phase, the elution order changes from toluene (sample solvent), C60, and C70 to C60, C70, and toluene at –50 °C (27 ; Figure 3). However, the change in elution order was not observed on the C18 diph phase, which A U G U S T 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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pected at lower temperatures, and therefore, the ligand distance becomes very important for interaction with C60. A weak interaction and a similar retention can be expected if there is any mismatch between the ligand spacing and the diameter of the C60 molecule. Solid-state NMR measurements with cross-polarization and magic-angle spinning support these results (29), thereby clearly indicating another contribution of fullerenes as sample probes, especially at low temperatures.
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With a microcolumn separation system, a limited amount of novel stationary phase can be easily evaluated. Fine, powdery, solid C60 was slurry-packed into a fused-silica capillary with cyclohexane as the 0 5 10 0 5 10 solvent, and the retention behavior of various aromatic compounds was systematically investigated in typical reversed-phase conditions (32). By careSolvent –70 °C –70 °C C70 ful interpretation of the retention behavior, it was C60 Solvent C60 C70 found that the solid C60 phase possessed unique shape selectivity toward isomeric PAHs; the solutes with a structure similar or partially similar to C60 0 5 10 0 5 10 were retained longer than other isomeric solutes. This trend is quite consistent with general obserRetention time (min) Retention time (min) vations in HPLC, especially for the retention behavior of aromatic analytes on aromatic bonded FIGURE 3. Chromatograms for the separation of C60 and C70 with (a) C18 and phases (5, 33). (b) C18 diph phases at various column temperatures. Chemically bonding C60 onto a silica support Mobile phase, n -hexane. (Adapted with permission from Ref. 31.) (34–37 ) significantly improved column performance, although the retention behavior for isomeric PAHs was very similar to that of solid C60 described has two bulky phenyl rings at the bottom of the bonded-phase earlier. Three calixarenes with different cavity dimensions, 4structure. No change in the elution order indicates that a con- tert-butylcalix[4]arene (TB4), 4-tert-butylcalix[6]arene (TB6), figurational change of the ODS phase as a function of temper- and 4-tert-butylcalix[8]arene (TB8), were separated with one ature is the most important factor influencing fullerene reten- of the chemically bonded C60 phases (36). The retention and tion. The elution order C60, C70, and toluene is caused by a kind selectivity for these three calixarenes were compared to those of of exclusion effect because the rigid alkyl chains do not have conventional ODS phases. Because the cavity diameter of TB8 enough space for the fullerene molecules to fit between adja- is ~1 nm and the diameter of C60 is
