Molecular Micelles:
Novel Pseudostationary Phases for CE
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Polymeric materials that can solvate compounds in much the same way as micelles provide an attractive alternative to conventional surfactant micelles as separation carriers.
Shahab A. Shamsi
ore and more, challenging analytical problems are driving the development and implementation of many new separation techniques. New stationary phases and methodologies that adequately separate very similar analytes are continuously being sought. Differences among analytes may be as apparent as the substituent position on a parent molecule or as subtle as chirality. A reagent’s ability to elucidate such distinctions is what ultimately contributes to its utility in a separation technique. We present a class of reagents, molecular micelles, which show considerable promise for separating a wide variety of analytes when used as pseudostationary phases in CE.
Georgia State University
Christopher P. Palmer
New Mexico Institute of Mining and Technology
Isiah M. Warner Louisiana State University
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In CE, the migration of analytes is influenced by the ratio of analyte charge to size (electrophoretic mobility) and the electroosmotic flow (EOF). The EOF is bulk solvent flow generated as a result of the applied electric field and the charge on the capillary interior wall (1). Because EOF affects all analytes and is often an order of magnitude larger than the electrophoretic mobility, analytes are typically separated in the following order: small cations, large cations, neutrals, large anions, and small anions. Although neutral molecules migrate with the EOF, they are not separated in the capillary. A variety of additives can alter the selectivity of CE separations. In 1984, Terabe showed that adding anionic surfactants to the buffer medium at a concentration sufficient to form micelles affects the separation of neutral analytes (2). Micelles are loosely defined as self-assemblies of surfactant molecules with a polar, often ionic, exterior and a lipophilic interior. Anionic micelles migrate through the buffer medium electrophoretically, similar to any other anionic compound. However, the migration time of a nonionic solute is determined by the extent of interaction with the migrating micellar phase. It should also be noted that the separation selectivity for ionic compounds can
Glossary (±)Alp BBMA (±)BNA (±)BNP (±)BOH GR GR-24 Poly SU Poly SUA Poly SUAA Poly SUAL Poly SUAV Poly SUL Poly SULA Poly SULL Poly SULV Poly SUV Poly SUVA Poly SUVL Poly SUVV Poly SUS (±)Prop
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(±)Alprenolol Butyl acrylate-butyl methacrylate-methacrylic acid (±)1, 1´-Bi-2-naphthyl-2,2´diamine (±)1, 1´-Bi-2-naphthyl-2,2´-diyl hydrogen phosphate (±)1,1´-Bi-2-naphthol Strigol analogues (2-Methyl-4-(2-oxo-2,3,3a,8b-tetrahydro-4H-indeno[1,2b]furan-3-ylidenemethoxy)-2but-2-en-4-olide) Poly(sodium undecylenate) Poly(sodium undecanoyl-L-alaninate) Poly(sodium undecanoyl-L-alanylalaninate) Poly(sodium undecanoyl-L-alanylleucinate) Poly(sodium undecanoyl-L-alanylvalinate) Poly(sodium undecanoyl-L-leucinate) Poly(sodium undecanoyl-L-leucylalaninate) Poly(sodium undecanoyl-L-leucylleucinate) Poly(sodium undecanoyl-L-leucylvalinate) Poly(sodium undecanoyl-L-valinate) Poly(sodium undecanoyl-L-valylalaninate) Poly(sodium undecanoyl-L-valylleucinate) Poly(sodium undecanoyl-L-valylvalinate) Poly(sodium undecenyl sulfate) (±)Propranolol
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be altered by interactions with the micellar phase. Terabe coined the term “micellar electrokinetic chromatography (MEKC)” for this approach. Although other acronyms have been suggested, we will use MEKC to be consistent with the inventor. The separation window in MEKC is determined by the solute that has the least interaction with the micelle (e.g., methanol elutes as an unretained component at time t0), and the most (e.g., Sudan III elutes at a micelle migration time tmc). The difference in migration time is then defined as EW = tmc – t0, with EW referred to as the elution window of the separation. Neutral molecules separated by MEKC will elute within this time window. Therefore, the larger the window, the more analytes can be included. Although they perform very well as separation carriers for many applications, micelles are limited in their applicability because they are dynamic structures in a state of equilibrium with free surfactant in the surrounding buffer. In addition, the micelle’s stability and structure are influenced by the structure and concentration of the surfactant and by properties of the buffer medium, such as pH, ionic strength, temperature, and organic modifier content. Polymeric materials (polysoaps, molecular micelles, micelle polymers) that can solvate compounds in much the same way as micelles provide an attractive alternative to conventional surfactant micelles as separation carriers. They can be synthesized by polymerizing surfactants with polymerizable groups or by copolymerizing monomers of both lipophilic and hydrophilic character to yield amphophilic copolymers. Dendrimers and modified dendrimers can also be used (3, 4). For our discussion, we include polymeric surfactants of three basic chemistries: T-type polymers synthesized in micellar form (Figures 1a, 1b, and 1i), linear copolymers (Figures 1c–1g), and dendrimers (Figure 1h). These polymeric phases, known as molecular micelles, have significant advantages as separation agents in CE and may provide a different solvation environment because of the steric constraints imposed by covalent stabilization of the polymer structure. Numerous studies and several reviews have been published on molecular micelles (5–10). The studies suggest that molecular micelles generally possess many properties similar to conventional nonpolymeric micelles.
Advantages of molecular micelles over CDs The most widely used chiral selectors in CE are cyclodextrins (CDs) and their charged and uncharged derivatives. These molecules are applicable to a wide variety of analytes for many reasons, one being that research on CDs and their derivatives
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as chiral separation agents has been ongoing for decades. In contrast, research on molecular micelles has been conducted for less than a decade. Despite their infancy, some advantages of molecular micelles over CDs have already surfaced. For example, the average degree of substitution for derivatized CDs varies over a range of 3–8, and, in many cases, the substitution is not accurately known (11, 12). Therefore, elucidating the chiral interaction mechanisms of derivatized CDs is sometimes difficult. However, the substitution problem is rapidly disappearing with the development of fully substituted CDs (13). Another advantage of molecular micelles over CDs is that different functionalities, including a variety of chiral headgroups, can be incorporated into polymers to offer a variety of selectivities. A third advantage is that amino acid-based polymeric surfactants bearing D and L optical configurations can easily be synthesized in high purity and characterized using a variety of analytical techniques (14–16). The availability of both the antipodes of a polymeric surfactant is particularly beneficial when it is necessary to reverse the migration order of two enantiomers to determine trace-level enantiomeric impurities with reasonable accuracy. One of the simplest examples of such an approach is the use of both antipodes to qualitatively profile the chiral discrimination of strigol analogues (so-called GR compounds), which infect cereals and legumes in sub-Saharan Africa and Asia (16, 17). One such compound, GR-24, contains four diastereomers with two enantiomeric pairs. As expected, when the D-form of the polymeric surfactant was replaced by the L-form, the migration order of the GR-24 enantiomers reversed. However, chiral resolution did not occur when the DL form was used; the GR-24 diastereomers were baseline-resolved instead. Thus, using the poly-DL surfactant confirmed that the synthesized GR-24 compound is indeed a mixture of two enantiomeric pairs (16). Polymeric surfactants have another major advantage over CDs in that parameters can be manipulated to provide various elution windows in EKC.
Advantages of polymeric surfactants Polymeric surfactants have several distinct advantages over conventional micelles. First, unlike conventional micelles, polymeric surfactants can be purified because they are covalently linked. Second, polymeric surfactants can be used below the critical micellar concentration (cmc) for the same reason. Third, polymeric surfactants are stable in the presence of inclusion molecules, such as CDs, and at relatively high concentrations of or-
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FIGURE 1. Structures of molecular micellar pseudostationary phases. (a) Poly(sodium undecylenate). (b) Poly(sodium undecenylsulfate). (c) BBMA. (d) Elvacite 2669. (e) Polyallylamine. (f) Acrylate copolymer. (g) Silicone polymer. (h) Modified dendrimer. (i) Amino acid-modified chiral micelle polymers.
ganic solvents. In contrast, organic solvents and inclusion compounds would tend to disrupt the formation of conventional micelles. Fourth, inclusion complexes such as cyclodextrins are more effective in combination with polymeric surfactants be-
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Time (min) FIGURE 2. Chromatograms of selected analytes using (a) octadecylmodified acrylate copolymers (Figure 1f) and (b) nonyl-modified polymers of the same structure. Changes in migration order indicate differences in selectivity. Peaks are 1, p-nitroaniline; 2, anisole; 3, nitrobenzene; 4, naphthalene methanol; 5, acenapthenol; 6, naphthyl amine; 7, naphthalene ethanol; 8, o-xylene; 9, naphthalene.
cause the polymers do not interfere with the formation of inclusion complexes between the analyte and the inclusion molecule. Finally, polymeric surfactants can have select properties fixed through the polymerization process. A disadvantage of polymeric surfactants and molecular micelles is that they must be synthesized, whereas conventional CDs are naturally occurring substances. However, most of the synthetic procedures are relatively easy to perform.
Achiral polymers Achiral polymeric materials used as pseudostationary phases extend the capabilities of EKC to separating hydrophobic compounds, introducing novel selectivity, and using MS detection. Separating hydrophobic compounds. The greater stability of polymeric phases results from eliminating the micellar equilibrium, which allows the use of high concentrations of organic modifiers. The organic modifiers facilitate the separation of hydrophobic compounds, such as polynuclear aromatic hydrocarbons (PAHs) and n-alkyl phenones, which would otherwise interact too strongly with the micelles and co-elute at tmc. Poly SU and poly SUS (Figures 1a and 1b) have been successfully used in buffers modified with up to 60% organic modifier to separate PAHs (18–20). Polyallylamine polymers (Figure 1e) have been used extensively to separate hydrophobic compounds in methanol-modified buffers (21). Separations of alkyl phenones using polyallylamines modified with alkyl chains of different chain lengths and buffers modified with up to 80%
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methanol have been reported. As the concentration of methanol increases, an initial broadening of the migration range occurs because of reduced electroosmotic mobility and increased polymeric surfactant mobility. Further increases in the methanol concentration reduce the resolution because of a decrease in interaction with the polymer. Another linear copolymer, Elvacite 2669 (Figure 1d), has also been used to separate hydrophobic compounds at concentrations of organic modifier in excess of 75% (22). Dendrimers modified with alkyl chains (Figure 1h) have also been used to separate hydrophobic compounds in buffers containing up to 90% methanol (23). Two recent studies with polyallylamine phases and Elvacite 2669 indicate that conformational change and intermolecular aggregation of the polymers take place at higher concentrations in aqueous media (24, 25). The structure and extent of polymer aggregation in solution depend on the organic modifier concentration. Changes in polymer conformation and aggregation correlate well with observed changes in electrophoretic mobility and chemical selectivity. Changes in chromatographic performance have also been observed with poly SU(18), poly SUS(19), and modified dendrimers (23), implying similar solution behavior. Polymer conformation and intermolecular aggregation are solvent-dependent and may introduce minor complications for using polymeric phases with organic modifiers, but they do not negate the fact that the polymers provide highly efficient and selective separations of hydrophobic compounds. Chemical selectivity. Another advantage of polymeric phases is that pseudostationary phases with very different chemical selectivity relative to conventional micelles can be created. Surfactants with substantial hydrophobic moieties are required to form micelles at low cmcs, which results in hydrophobic interactions being one of the dominant factors in determining the retention and selectivity of micellar phases. With polymeric phases, the requirement of self-association is eliminated, and polymers with varied structures can be used. Often, very different chemical selectivity is observed between polymeric phases and micelles, and among different polymeric phases. For example, linear acrylate copolymers with varied side chain chemistries vary significantly in their chemical selectivities. The chromatograms in Figure 2 demonstrate the different selectivities for polymers modified with octadecyl versus nonyl chains. Predictably, hydrophobic compounds interact more strongly with the polymers modified with octadecyl chains. Significantly, the separation selectivity can be adjusted in a predictable way by using mixtures of polymers with different se-
lectivities (26). This approach will become more useful as families of polymers with different chemical selectivities based on side group chemistry can be developed. Other polymeric phases have demonstrated even greater differences in chemical selectivity. A pseudostationary phase based on a silicone backbone (Figure 1g) provides very different selectivity from sodium dodecyl sulfate (SDS) micelles (27). A polymeric dye provides very different selectivity relative to SDS micelles for the separation of aromatic compounds (28). Polyethyleneimine phases demonstrate unique selectivity for separating phenolic compounds (29). Unmodified dendrimers separate compounds based on the backbone structure (e.g., benzene vs naphthalene) rather than hydrophobicity (30). No hydrophobic selectivity is observed. Modification of the dendrimers with alkyl groups of increasing chain length changes the selectivity from a separation based on backbone structure to one more similar to SDS micelles. The variety of polymeric phases that can be used as carriers in EKC has only just begun to be explored. As more polymeric structures are investigated, the range of chemical selectivities and separation problems that can be solved will increase. Fundamental factors contributing to chemical selectivity in polymeric phases are also being investigated and will lead to better chemical separations and improved phases. MS detection. Molecular micelles can be used in combination with MS detection. Conventional micellar systems are difficult to use with MS detection because the high concentration and surface activity of low molecular weight surfactants interfere with the electrospray ionization process. The primary advantage of polymeric surfactants for this application is their high molecular weight. Except in the case of high degrees of ionization, the polymeric surfactants do not interfere with detecting analytes of significantly lower molecular weight than the polymeric surfactant. Polymeric phases can also be used at lower concentrations, sometimes even below the cmc of similar conventional surfactants, making them less likely to interfere with electrospray ionization and detection. Ozaki et al. first demonstrated this principle using a linear acrylic acid copolymer (BBMA, Figure 1c) and four analytes (31). Lu et al. have also demonstrated the concept using an SUS polymer and electrospray ionization MS to separate and detect tricyclic antidepressant and -adrenergic blocker drugs (32).
Chiral polymeric surfactants A primary advantage of chiral polymeric surfactants over natu-
rally occurring chiral selectors (e.g., CDs) is that chiral selectivity can be manipulated. The synthesis of a T-type chiral polymeric surfactant, N-undecanoyl-L-valinate, was published in 1994 (33). Another publication from the same laboratory provided details on the synthetic methodology to obtain several polymeric N-undecanoyl-L-amino acid and N-undecanoyl-Ldipeptide derivatives (14). It should be noted that some patents have also been reported on polymeric surfactants (34, 35).
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FIGURE 3. Comparison between conventional, unpolymerized micelle and polymeric surfactant for separation of (±)BOH. (a) 0.5% (w/v) L-SUV; current is 40 mA. (b) 1% (w/v) L-SUV; 51 µA. (c) 0.05% (w/v) poly SUV; 30 mA. (d) 0.5% poly SUV; 39 µA. Chiral MEKC conditions: 25 mM borate, pH 9.0, applied voltage is 12 kV, UV detection at 290 nm.
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Single amino acid polymeric surfactants. Wang and Warner and Dobashi et al. were among the first to report the use of a single amino acid polymeric surfactant as a chiral pseudostationary phase for EKC (33, 36). Wang and Warner demonstrated numerous advantages of polymeric surfactants over conventional nonpolymerized micelles, while Dobashi provided a detailed comparison to conventional micelles. Figure 3 compares the chiral recognition ability of the polymeric surfactant poly SUV and its corresponding nonpolymerized micelle L-SUV for enantiomeric separation of the atropisomers (±)BOH. Two points of interest emerge from this comparison. First, note that when using 0.5% (w/v) L-SUV, no chiral separation of (±)BOH was obtained, because this is below the cmc of L-SUV micelle (Figure 3a). However, poly SUV provided baseline resolution of (±)BOH even when the concentration of the polymer was as low as 0.05% (w/v), which is below the cmc of the monomers (Figure 3c). Second, it is clear that a nonpolymerized micelle L-SUV also provides chiral separation, but this occurs only at a higher concentration [i.e., 1% (w/v)] with a longer migration time and much lower efficiency (Figure 3b). The average plate number of (±)BOH was 102,240 using poly SUV (Figure 3d) compared with only 28,070 obtained with LSUV (Figure 3b). Therefore, this polymeric surfactant allows better chiral discrimination over its nonpolymerized counterpart. The results obtained by Wang and Warner were found to be consistent with the spectroscopic data by Paleos and co-workers (37 ). On the basis of these observations, Wang proposed that the solute does not penetrate as deeply into the core of the polymeric surfactant as it does in the case of normal micelles (33). Therefore, it is reasonable to expect a faster rate of solute mass transfer into and out of the polymeric surfactant. The work of Dobashi’s and Warner’s groups has extended the applicability of this polymeric system for chiral separations (3, 36). Dipeptide polymeric surfactants. Because poly SUV provided such a fruitful approach to enantiomeric separation, a chiral dipeptide polymer with multifunctional chiral binding sites was the next obvious choice. Shamsi and co-workers initiated a systematic process toward tackling this task (38). The dipeptides poly SUAA, poly SUVV, and poly SULL differ from poly SUA, poly SUV, and poly SUL by one additional amino acid group attached to the hydrocarbon chain (Figure 1i). Therefore, poly SUAA, poly SUVV, and poly SULL are dipeptide-terminated polymeric surfactants in which the carbonyl group of one amino acid is attached to the amino group of another amino acid by a peptide bond. Further, the dipeptide surfactants not 146 A
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Molecular micelles have significant advantages as separation agents in CE and may provide a different solvation environment.
only have two chiral centers but also two additional hydrogen-bonding sites of the same optical configuration. Several examples of improved chiral resolutions with decreased migration times of cationic racemates (e.g., (±)Prop and (±)Alp) are shown in Figure 4. Although generalizations are difficult to make in chiral separations, some observations are noteworthy. The enantiomers of Alp and Prop eluted in order of increasing hydrophobicity for both polymeric surfactants. In addition, the (S)-(-) enantiomer of each racemate always eluted before the (R)-(+) form. Therefore, the migration time and elution order appear to be a direct consequence of analyte–polymeric surfactant binding. Moreover, comparing Figures 4a versus 4b and 4c versus 4d clearly illustrates that an increase in elution time for both (±)Alp and (±)Prop using single amino acid surfactants does not lead to any enhancement in chiral resolution Rs. This suggests that the improved chiral separation of these cationic racemates using dipeptide polymeric surfactants is controlled by steric factors rather than by the hydrophobicity of the chiral pseudostationary phase. It is plausible to conclude that an increase in the number of stereogenic centers and hydrogenbonding sites on the polar head group of poly SUAA and poly SUVV contributed to this superior chiral discrimination over the respective poly SUA and poly SUV polymeric surfactants. Amino acid order. The improved chiral separations obtained by using dipeptide polymeric surfactants encouraged Billiot and co-workers to attempt to understand these chiral separations. They synthesized various polymeric dipeptide surfactants with different chiral centers, combinations, and configurations. They first studied, in detail, the effect of the order of amino acids in the dipeptide surfactants on chiral separation (39). The two main dipeptide surfactants used were poly SULV and poly SUVL. The difference in chiral Rs of (±)BNP using these two dipeptide surfactants was very pronounced. The enantiomers of BNP were resolved using poly SULV with a very large Rs value of 8.0, while an Rs of
