Anal. Chem. 1995, 67, 1722-1726
Resorcarenes as Pseudostationary Phases with Selectivity for Electrokinetic Chromatography Knut B€ichmann,* Alexis Bazzanella, lngo Haag, and KwaneYong Han Technische Hochschule Darmstadt, Fachbereich Chemie, Petersenstrasse 18, 64287 Darmstadt, Germany
Ralf Amecke, Volker Btbhmer, and Walter Vogt Johannes-Gutenberg-Universitat, Fachbereich Chemie, J.J.-Beeher-Weg 18-20, 55099 Mainz, Germany
Resorcarenes, macrocyclic molecules built up by four alkylidene-bridgedresorcinol units, have been studied as new pseudostationary phases in order to develop a different mode of pseudophase and to attain a distinct selectivityfor electrokineticchromatography (EKC). This novel pseudostationaryphase has several advantages over traditionally used surfactants in micellar electrokinetic chromatography. First, the stable structure of the cyclic tetramer permits the use of high contents of organic (v/v) acetonitrile, to adjust optimum modifiers, i.e., >60% capacity factors. Second, high electrophoreticmobility of the resorcarenes is based on four negative charges delocalized and stabilized over hydrogen bonds. This supplies a broad elution range, which is a main parameter for resolution of separated peaks. Furthermore, these resorcarenes possess unique selectivity for hydrophobic compounds. The order of peak elution with a test mixture involving 12 polycyclic aromatic hydrocarbons (PAHs)is almost identical with that from reverse-phase highperformance liquid chromatography. The kst two terms exclude the contribution of microheterogeneity that decreases the efficiency of EKC when common surfactants are used as pseudophases. The selectivities of resorcarenes with methyl, pentyl, undecyl, and p-chlorophenyl groups at the bridging carbons in separation of PAHs are discussed as a function of these residues. A number of approaches have been shown to enhance the ability and the selectivity of capillary electrophoresis (CE) to analyze uncharged species. Since the early studies of Terabe et a1.l with a micellar phase in 1984, various organic molecular discrete molecules?-" or chromatographic particles12 have been employed as an additional phase-a pseudc(1) Terabe, S.; Otsuka, K; Ichikawa, K; Tsuchiya, A; Ando, T. Anal. Chem. 1984,56,111-113. (2) Dobashi, A; Ono, T.; Hara, S.; Yamagushi, J. J. Chromatogr. 1989,480, 413-420. (3) Cole, R 0.;Sepaniak, M. J.; Hme, W. L.; Gorse, J.; Oldiges, K J. Chromatogr, 1991,557,113-123. (4) El Rassi, Z.; Cai, J. J. Chromatogr. 1992,608,31-45. (5) Watarai, H.; Ogawa, IC;Monta, T.; Takahashi, I. Anal. Sci. 1991,Suppl. 7, 245-248. (6)Terabe, S.; Matsubara, N.; Ishihama, Y.; Okada, Y. j . Chromatogr. 1992, 608,23-29. (7) Aiken, J. H.; Huie, C. W. Chromatographia 1993,35, 448-450. (8) Terabe, S.; Miyashita, Y.; Shibata, 0.; Barnhart, E. R; Alexander, L. R; Patterson, D. G.; Karger, B.; Hosoya, K; Tanaka, N. J. Chromatogr. 1990, 516,23-31.
1722 Analytical Chemistry, Vol. 67, No. 10, May 75,7995
b:R = CH,
Calix[4]arene
&:R = C,H,, &:R = C,,H,, 1p:R = p-chlorophenyl Figure I.Studied pseudostationary phases.
stationary phase-in CE for the same purpose. This offers the possibility for neutral species to be distributed between the added phase and the electrolyte with distinct partition coefficients. Several investigations were performed using various types of pseudostationary phase, such as micelles in micellar electrokinetic chromatography (MEKC)2,3,4 or microemulsions in microemulsion electrokinetic chromatography (MEEKC),5,6,7 In MEKC, the separation of highly hydrophobic analytes, i.e., polycyclic aromatic hydrocarbons (PAHs),with aqueous electrolytes is generally impossiblebecause of their large capacity factors due to the extreme tendency of the partitioning toward the micellar phase.s Macrocyclic molecules, Le., cyclode~trins~~~ or crown ethers,l0were added to the micellar media to supply another equilibrlum distribution. Owing to the limited stability of micellar phases, however, the content of organic solvent cannot be increased to an amount sufficient to dissolve the hydrophobic analytes. In MEEKC, the solubility can be enhanced, compared with MEKC, by changing the composition of the microemulsions. However, the absence of various detailed compositional investigations for stable and suitable microemulsions is a main obstacle in MEEKC. Resorcarenes (Figure 1) are cyclic tetramers which are easily obtained by acidcatalyzed condensation of resorcinol with a large (9) Yik, Y. F.; Ong, C. P.; Khoo, S. B.; Lee, H. IC; Li, S. F. Y. J. Chromatogr. 1992,589,333-338. (10) Kuhn, R; Stoecklin, F.; Erni, F. Chromatographia 1992,33, 32-35. (11) Shohat, D.; Gmshka, E. Anal. Chem. 1994,66,747-750. (12) Bachmann, K; Gottlicher, B.; Haag, I.; Han, IC-Y.; Hensel, W.; Mainka, A J. Chromatogr. 1994,688,283-293. 0003-2700/95/0367-1722$9.00/0 0 1995 American Chemical Society
R
7
14-
Figure 2. Tetraanion of a resorcarene.
variety of aldehydes (other than formaldehyde).13J4 They are available in one step in large quantities, and usually exclusively the all-cis stereoisomer is formed. It assumes a cone conformation with an axial orientation of all residues R stabilized by intramolecular hydrogen bonds between OH groups of adjacent resorcinol units. Four protons are easily removed to form a C~symmetrical tetraanion stabilized by four hydrogen bonds of the type A-0-Ha a-Ar (Figure 2) . 1 5 3 Potentiometric titrations in water and mixed solvents show pK values for these four protons lower than those of resorcinol by 2 units.15 The structure of resorcarenes has several essential merits for its employment in EKC. From the perspective of chromatography, the residue R (see Figure 1) can be modified for the corresponding chromatographic purpose. In other words, the investigation of different selectivities of a pseudostationary phase with identical charge density is possible. Due to the stability of the resorcarene molecule, the content of organic solvent can be varied to any extent desired. The stability of the pseudostationary phase is, therefore, no longer limiting factor, and the content of organic solvent can be freely adjusted for optimum capacity factors (k'). The electrophoretic mobility of a resorcarene is based on its tetraanion. This defined structure with a uniform charge excludes microheterogeneity, which is one of the dominant factors that contributes to bandbroadening in MEKC." In this paper, the first results of successful separations of 12 PAHs with resorcarenes as a pseudostationaryphase in EKC are reported. EXPERIMENTAL SECTION
Syntheses. To a stirred solution of 0.1 mol of resorcinol in 60 mL of ethanol and 20 mL of concentrated hydrochloric acid at 5 "C was added 0.1 mol of aldehyde dropwise over 1 h. A precipitate formed which dissolved again on heating. The solution was refluxed for 8 h and then cooled to room temperature. A gummy precipitate formed, which became crystalline upon addition of water. Filtration, washing with water, and recrystallization from acetone gave the pure resorcarene.13J4 Apparatus. The electrokinetic experiments were carried out on a laboratory-built CE system that has been described in detail elsewhere.ls A modification was the detection performed in the UV mode operating at 260 nm @ionex, Idstein, Germany). Untreated fused-silica capillaries with an inside diameter of 50 pm (13) Sverker Hogberg, A G. J.Am. Chem. SOC.1980, 102, 6046-6050. (14) Sverker, Hogberg, A G. J. 0%.Chem. 1980,45,4498-4500. (15) Schneider, H.-J.;Giittes, D.; Schneider, U. J. Am. Chem. SOC.1988, 110, 6449-6454. (16) Schneider, H.-J.;Theis, I. Angew. Chem. 1991, 103, 1419-1439. (17) Terabe, S.; Otsuka, IC;Ando, T. Anal. Chem. 1989, 61, 251-260. (18) Btichmann, IC; Boden, J.: Haumann, I. J. Chromatogr. 1992, 626, 259265.
Figure 3. UV spectrum of resorcarene I C in electrolyte (compare Figure 8).
were employed (ChromatographicService, Langewehr, Germany). The total length of the capillary varied from 70 to 80 cm. Sample injection was accomplished by the hydrostatic method. Reagents, HPLC grade acetonitrile was obtained from Fluka (Neu-Ulm, Germany). A stock solution of test analytes, PAHs (Aldrich, Steinheim, Germany), was prepared at a concentration of approximately 500 ppm. This solution was diluted further with running electrolyte consisting of an acetonitrile-water hydroorganic phase and urea (Fluka) at a concentration stated in the text. Water was deionized with a combination of Milli-RO plus 10 and Milli-Q systems (Millipore, Eschbom, Germany). All electrolytes were filtered through a 0.22 pm filter (Millipore). All chemicals and solvents were used as obtained. Procedures. A region of the polyimide coating was removed to generate a transparent detection "window" at 14 cm from the outlet of the capillary. The capillaries were rinsed with HzO, HCl (1 M), HzO, NaOH (1 M), HzO, and the running buffer each for 5 min. These rinse cycles were performed every day before the first run and whenever the buffer solution was changed. Before each injection, the capillaries were rinsed with running buffer for 5 min. In the evening, the capillaries were rinsed with water for 20 min and then dried with air. Hydrostatic sample injections were manually performed by replacing the inlet reservoir with sample vial and raising it 10 cm above the outlet end for 30 s. W Absorptivity of Resorcarenes. Figure 3 shows the UV spectrum of resorcarene IC in the electrolyte used for the separations (compare Figure 8). A wavelength of 260 nm was determined to be most favorable for UV detection of the PAHs. RESULTS AND DISCUSSION
Electrophoretic Characterization. The mobilities of the resorcarenes for electrophoretic characterization of these s u b stances were determined using a reiterative procedure first described by Bushey and Jorgenson for MEKC based upon a homologous series.lg The plot of log k' versus the carbon number for a homologous series must yield a straight line. Assuming the retention time of the last eluting substance temporarily as the migration time of the macromolecule, t ~it is , possible to calculate k' for all separated homologous by (19) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1989, 61, 260-264.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1723
log k’
log k’ is plotted versus the carbon number, and a linear fit is employed. The k’ value for the last eluting homologue is found from the plot, and a new t~ is obtained from eq 1. All k’ values are recalculated, and the procedure is reiterated until the most linear plot is yielded and t~ converges to a unique value. A modification of this method20 based upon PAHs uses the linear correlation between log k’ and the so-called correlation factor, F, which replaces the carbon number for homologous series for the linear correlation. The correlation factor F1J2 is calculated by
-IC
-Id -c
-0,5
-‘5
k’ = Keq@
6
7
8
9
10
11
correlation factor F Figure 4. Linear correlation between log K of PAHs and correlation and Id. factor Ffor resorcarenes l b , IC,
F = no. of double bonds +. no. of primary and secondary carbons 0.5 for a nonaromatic ring (2) Using this procedure, the t~ values and the mobility for the resorcarenes l b , IC,and Id were determined to be 67.3, 48.7, and 53.2 min, respectively. The linear fits obtained from the final t~ values are presented in Figure 4. Efficiency. In Figure 5, the dependence of the plate height, H, on the electroosmotic velocity, u ~is ~shown , for three PAHs with different capacity factors and resorcarene ICas pseudostationary phase. Optimum results were obtained for applying a voltage of 20 kV, which leads to plate heights between 3.6 and 5.4 pm. Theoretical plate numbers obtained are in the range of 140 000-320 000. S i l a r results were found for all resorcarenes employed. Higher voltages lead to peak broadening due to increased Joule heating. Variation of Capacity Factors. The capacity factor is determined from the distribution equilibrium (Keq)of the analyte between the two phases and by the phase ratio CP:
~
lb
8
0
I\ I
I
3
4
5
8
7
8
9
1
u(w) [cm/mln] Figure 5. Dependence of the plate height H on the linear velocity of the electroosmotic flow. Electrolytescontained 10 mM resorcarene IC, 6 M urea, and 50% (vh) acetonitrile at pH = 13.25. ANT, anthracene; PHE, phenanthrene; and BaP, benzo[a]pyrene.
k 4
(3)
7 * CHR
* EaP
In conventional chromatography, the phase ratio is a k e d property of the selected column, and capacity factors can be varied only by changing the eluting strength of the mobile phase. In EKC, however, it is easy to modify the phase ratio by changing the concentration of the surfactant or the pseudostationary phase. Capacity factors are found to be linearly correlated with this c o n ~ e n t r a t i o n .Figure ~ ~ ~ ~ ~6 shows plots of k’ versus the concentration of resorcarene ICfor some PAHs, and the expected linear relationship is obtained. Obviously, the slopes of the linear plots increase with the hydrophobicities of the different PAHs, due to stronger dependence of the capacity factor from the phase ratio with increasing hydrophobicity of the analyte. Influence of Host-Guest Interactions. One important question, which cannot be overlooked within the complete characterization of a new pseudostationary phase, concerns the (20) Palmer, C.P.; Khaled, M. Y.; McNair, H. M. J. High Resolut. Chromatogr. 1992, 15, 756-762. (21) Schabron,J. F.; Hurtubise, R J.; Silver, H. F. Anal. Chem. 1977,49,22532260. (22) Hurtubise, R. J.; Allen, T.W.; Silver, H. F. J. Chromatogr. 1982,235, 517522. (23) Terabe, S.; Otsuka, IC; Ando, T. Anal. Chem. 1985, 57, 834-841. (24) Saitoh, IC;Kiyohara, C.; Suzuki, N. J. High Resolut. Chromatogr. 1991,14, 245-248.
1724 Analytical Chemistty, Vol. 67, No. 10, May 15, 1995
4 5 6 7 8 0 1 0 1 1 1 2 concentration of resorcarene [mMl Figure 6. Influence of resorcarene IC concentration on capacity factors of PAHs. A linear dependence is observed. Electrolytes contained 6 M urea and 50% (vh) acetonitrile at pH = 13.25. NAP, naphthalene;PHE, phenanthrene; FLU, fluoranthene; CHR, chrysene; and BaP, benzo[a]pyrene.
3
kind of interaction between the analyte and the pseudophase. Rewrcarenes as well as the closely related calix[4larenes are [1.1.1.11metacyclophanes, assuming the so-called cone conformation (compare Figure 2). The cavity thus formed possesses pronounced hydrophobic character in the case of calixarenes, where the four hydroxyl groups in the endo positions form a “closed” circle of intramolecular hydrogen bonds. Various host-guest complexes of calixarenes with hydrophobic guests like neutral arenes and PAHs are kn0wn.25826The driving forces for these host-guest associations are hydrophobic in nature, van der Waals and CH3-7~ interactions. Resorcarenes, on the other hand, have (25) Gutsche, C. D.; Nam,IC C.J. Am. Chem. SOC.1988,110, 6153-6162. (26) Gutsche, C.D.; Alam,I. Tetrahedron 1988, 44, 4689-4694.
14000
12000
a)
10000 80000-’ 60000--
10000
40000--
80000
I
20000-~
60000
I
I ’
21
22
23
24
25
26
lminl
5
10
15
20
25
30
[mini
Flgure 8. Separation of 12 PAHs with resorcarene IC. Condi50% (v/v) acetonitrile, and 6 M urea, tions: electrolyte, 5.8 mM IC, pH = 13.25; capillary, length 76 cm (62 cm to detector), i.d. 50 pm; injection, hydrostatic 10 cm, 30 s;detection, UV at 260 nm; voltage, 20 kV; current, 6 pA. Peak identification: (1) naphthalene, (2) phenanthrene, (3) anthracene, (4) fluoranthene, (5) pyrene, (6) triphenylene, (7) chrysene, (8) benzo[qfluoranthene, (9) benzo[a]pyrene, (10) indeno[l,2,3-cd]pyrene, (11) anthanthrene, and (12) benzo[gh/]perylene.
Figure 7. Investigation of the effect of the host-guest interaction on the separation of PAHs with resorcarene l a and calix[4]arene (see Figure 2). (a) Separation of PAHs with resorcarene l a . Conditions are as in Figure 8. (b) Separation of PAHs with calix[4]arene. Conditions: electrolyte, 10 mM calix[4]arene, 50% (v/v) acetonitrile, and 6 M urea, pH = 12.7; capillary, length 80 cm (65 cm to detector), i.d. 50 pm; injection, hydrostatic 10 cm, 30 s; detection, UV at 260 nm; voltage, 20 kV; current, 5 PA.
eight hydroxyl groups in the ex0 positions, formkg four hydrogenbonded pairs. Host-guest interactions with dicarboxylic di01s,~-~ and ~ u g a r s ~ O mainly - ~ ~ occur via further hydrogen bonds. However, interactions with the hydrophobic center of the cavity remain important, as demonstrated by the complexationof various “monools”. In order to examine the contribution of host-guest interactions to the retention of PAHs with rewrcarenes as the pseudostationary phase, we investigated a series of resorcarenes with different residues R In comparison to the methyl-substitutedresorcarene la, we studied also the unsubstituted calix[4larene (see Figure 11, which possesses a similar cavity. (The unsubstituted resor(27)Tanaka, Y.;Kato, Y.;Aoyama, Y .J. Am. Chem. SOC.1990,112,2807-2808. (28)Kikuchi, Y.;Tanaka, Y.; Kato, Y.;Aoyama, Y./.Am. Chem. SOC.1991,113, 1349-1354. (29)Kikuchi, Y.; Kobayashi, IC; Aoyama, Y.J.Am. Chem. SOC.1992,114,13511358. (30)Tanaka, Y.;Sugahara, S.; Aoyama, Y.J.Am. Chem. SOC.1989,111,53975404. (31)Tanaka, Y.;Ubakata, Y.; Aoyama, Y. Chem. Lett. 1989, 1905-1908. (32)Kobayashi, IC;Tominaya, M.; Asakawa, Y.;Aoyama, Y . Tetrahedron Lett. 1993,34,5121-5124.
carene with methylene bridges is not easily available.33 But due to the absence of the axial substituent R, it would be much more flexible than usual resorcarenes and therefore not unambiguously comparable.) Both substances were employed as pseudostationary phases under the normal conditions. Figure 7a shows the attempt to separate 12 PAHs with methyl-substitutedresorcarene, and Figure 7b shows the same separation using unsubstituted calixl4larene. Both electropherograms are rather similar, and both systems obviously show only poor selectivity and capacity factors near zero. Due to these experimental results, the participation of host-guest interaction between PAHs and the negatively charged macrocycle can be assumed to be negligible in the retention process. Thus, obviously the alkyl chains in resorcarene l b and IC and the aromatic substituents in resorcarene Id substantially contribute to the retention process, whereas the macrocycle mainly serves as a “platform”,carrying and stabilizing the charges and defining the distance between the substituents. In a way, the resorcarene molecule corresponds to the stationary phase in reverse-phase high-performance liquid chromatography (RP-HPLC). The macrocycle replaces the silica particle as carrier for the hydrophobic alkyl or aryl substituents having the RP effect. This analogy results in a nearly identical retention behavior of PAHs in EKC with resorcarenes compared to RP-HPLC.34,35 Figure 8 shows the separation of 12 PAHs with resorcarene IC. The elution order of these components corresponds to the order of F numbers21e and the L/B which are common parameters in the reversed-phase mode to correlate the retention behavior of PAHs with their molecular size, shape, and hydro(33)Falana, 0.M.; AI-Farhan, E.; Keehn, P.M.; Stevenson, R Tetrahedron Lett. 1994,35,65-68. (34)May, W.E.;Wise, S. A Anal. Chem. 1984,56, 225-232. (35)Chen, J.; Steenackers, D.; Medvedovici, A; Sandra, P. J. High Resolut. Chromatogr. 1993, 16,605-608. (36)Kaliszan, R;Lamparczyk, H. J. Chromatogr. Sci. 1978, 16, 246-248. (37)Wise, S.A;Bonnett, W. J.; Guenther, F. R; May, W. P. J. Chromatogr. Sci. 1981,19,457-465.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1725
IrVl
-
4 0 0 0 0 0-
495
2'6 2'8 3'0 3'2 3'4 3'6 [ m i n l Figure 9. Separation of 12 PAHs with resorcarene l b . Conditions: electrolyte, 12.8 mM I b ; current, 9 PA; other conditions and peak identification as in Figure 8.
400000
4
Bji
11,12
1OOOOO
18
m
22
M
26
za
M
[mini
Figure 10. Separation of 12 PAHs with resorcarene Id. Conditions: electrolyte, 6.4 mM I d ; current, 5 PA; other conditions and peak identification as in Figure 8.
phobicity. The only difference in the elution sequence is found for indeno [1,2,%cd]pyreneeluting before its isomer benzo [ghilperylene in EKC, vice versa in W-HPLC. These isomers are most d ~ c u lto t separate on octadecylsilane (ODS) stationary phases but were easily separated with resorcarene IC. The separation of a third isomer, anthanthrene, from the other two demonstrates the great selectivity of this pseudostationary phase. Various Selectivities. Figures 9 and 10 show the separations of a mixture of 12 PAHs with resorcarene l b and Id. To examine the selectivity of the different resorcarene derivatives, the used PAH mixture contains several pairs of isomers, Le., phenanthrene/ anthracene, fluoranthene/pyrene, chrysene/triphenylene, benzo-
1726 Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
[k]fluoranthene/benzo[alpyrene, and indeno[l,2,3-~dIpyrene/ anthanthrene/benzo [ghilperylene. Comparing the three resorcarene derivatives investigated, only the resorcarene with the longest alkyl chain, IC,shows appropriate selectivity to separate all 12 PAH components (Figure 8). Using resorcarene l b as pseudostationary phase, coelution of phenanthrene/anthracene, fluoranthene/pyrene, and indeno [ 1,2,3-cd]pyrene/anthanthrene/ benzo [ghi]perylene was obtained. Even with higher concentrations of lb, separation of these isomers was not achieved. Consequently, not only is the carbon content in the electrolyte solution accountable for the separation power of resorcarenes, but also different chain lengths cause different selectivities. The much greater hydrophobicity and flexibility of a C11 compared with a CS chain should be responsible for this fact. Figure 10 shows the same separation with resorcarene Id. In this case, n-a interactionsbetween the PAHs and the p-chlorophenylgroups are used for separation. Isomers like phenanthrene/anthracene and anthanthrene/benzo [ghilperylene coelute, baseline and resolution is not possible for several PAHs. CONCLUSIONS
The employment of resorcarenes easily synthesized from resorcinol and various aldehydes as a novel pseudostationary phase in EKC led to an entirely new separation mode. The high content of organic solvent and the reverse-phase-like selectivity as well as the high mobility due to formation of a tetraanion have allowed the separation of PAHs with molecular weights higher than 200. The resorcarene molecule may be varied in numerous ways, either by use of different aldehydes during the synthesis or by subsequent introduction of additional functional groups, in order to achieve various distinct selectivities. We are presently studying these possibilities over a broader range. Our special interest is focused on the relationship between the retention of several analytes and the number, distance, and length of alkyl chains attached to the basic macrocycle, and on the influence of the ratio between hydrophobicity and charge density on the selectivity of the separation. In order to examine retention mechanisms, resorcarenes could be an important linkage between EKC and other chromatographic methods like RPHPLC. In future papers, studies about various charomatographic and/or electrophoretic features of resorcarenes will be discussed. Received for review October 5, 1994. Accepted March 6, 1995.a AC9409804 @
Abstract published in Advance ACS Abstracts, April 1, 1995.
