Design of Chromatographic and Electrophoretic Separation Based on

Ion-pair formation of polyammonium ions with aromatic disulfonates (ADSs) has been investigated with ion-exchange chromatography. Data obtained imply ...
0 downloads 0 Views 265KB Size
Download PDF
Anal. Chem. 1996, 68, 1158-1163

Design of Chromatographic and Electrophoretic Separation Based on Ion-Pair Formation of Aromatic Disulfonates with Polyammonium Ions Tetsuo Okada

Department of Chemistry, Faculty of Science, Tokyo Institute of Technology, Meguro-ku, Tokyo 152, Japan

Ion-pair formation of polyammonium ions with aromatic disulfonates (ADSs) has been investigated with ionexchange chromatography. Data obtained imply that polyethyleneammonium ions [NH3+(CH2CH2NH2+)nH] recognize the lengths between sulfonate groups in ADSs in the absence of specific ion-pair formation. The molecular length of the diethylenetriammonium ion is almost equal to that between sulfonate groups in ADSs tested when this polyammonium ion adopts an all-trans conformation; the match of lengths dominates ion-pair formation. However, anthraquinone-1,8-disulfonate (1,8-AnDS) shows a different behavior. This compound specifically interacts with polyammonium ions having an ethylenediammonium structure in a molecular terminal and forms much more stable ion-pairs than any other ADS. It is speculated that the accommodation of a terminal ammonium ion in the pocket consisting of two sulfonate groups in the 1,8-AnDS molecule allows stable complex formation. These are applied to the enhancement of the selectivity in chromatographic and electrophoretic separation of ADS. Separation occupies an important and often substantial part in experimental approaches to both fundamental and practical chemistry. More selective, more specific, and more effective separation is required as samples dealt with in research become more complex. The need to develop separations bearing novel features has turned the attention to host-guest or supramolecular chemistry. Crown ethers and cryptands are representatives of synthesized hosts acting as Lewis bases, and they are extensively used in the separation of cations.1-9 In contrast, despite much effort, efficient compounds complexing anions (or Lewis bases) are very few. Polycations have been studied as candidates for Lewis acids (1) Inoue, Y., Gokel, G. W., Eds. Cation Binding by Macrocycles: Complexation of Cationic Species by Crown Ethers; Marcel Dekker: New York, 1990. (2) Izatt, R. M., Christensen, J. J., Eds. Synthetic Multidentate Macrocyclic Compounds; Academic Press: New York, 1978. (3) Fenton, D. E. In Comprehensive Coordination Chemistry; The Synthesis, Properties and Applications of Coordination compounds; Wilkinson, G., Gillard, R. D., McCleverty, J. A., Eds.; Pergamon: Oxford, 1987; Vol. 3, Chapter 23. (4) Poonia, N. S.; Bajaj, A. V. Chem. Rev. 1979, 79, 389-445. (5) An, H.; Bradshaw, J. S.; Izatt, R. M.; Yan, Z. Chem. Rev. 1994, 94, 939991. (6) Troxler, L.; Wipff, G. J. Am. Chem. Soc. 1994, 116, 1468-1480. (7) Visser, H.; Reinhoudt, D. N.; de Jong, F. Chem. Soc. Rev. 1994, 75-81. (8) Takeda, Y.; Kawarabayashi, A.; Takahashi, K.; Kudo, Y. Bull. Chem. Soc. Jpn. 1995, 68, 1309-1314. (9) Tsurubou, S.; Mizutani, M.; Kadota, Y.; Yamamoto, T.; Umetani, S.; Sasaki, T.; Le, Q. T. H.; Matsui, M. Anal. Chem. 1995, 67, 1465-1469.

1158 Analytical Chemistry, Vol. 68, No. 7, April 1, 1996

capable of recognizing multicharged Lewis bases.10-14 A number of chemists have focused their attention mainly on cyclic polyammonium ions and succeeded to synthesize some compounds showing specific complex formation abilities.12-14 They have been devoted to developing such complicated hosts because many compounds have been successfully designed according to the sizefit concept,15,16 which is often mistakenly used as if it were the only mechanism governing the selectivity in host-guest chemistry, where a host accommodates a guest in its cavity. The sizefit is not the only one but is one of the most important mechanisms, and therefore even an acyclic host may show specificity or selectivity; it can be a problem that molecular flexibility of acyclic hosts complicates the interpretation of results. In this paper, we present the ion-pair formation behaviors of polyammonium ions (PAs) with aromatic disulfonic acids (ADSs) and describe the function of PAs as acyclic hosts for ADSs and designs of the novel separation of ADSs using PAs. PAs having various chain lengths are commercially available and are expected to act as a molecular ruler, recognizing the structures of ADSs. EXPERIMENTAL SECTION The chromatographic system was composed of a computercontrolled pump model CCPD or CCPM (Tosoh), a Rheodyne injection valve equipped with a 100 µL sample loop, a UV detector (Model 875-UV, JASCO), and a strip-chart recorder. A separation column was immersed in thermostated water to keep the temperature constant at 25 °C, unless otherwise stated. The separation column used to determine the ion-pair formation constants of ADSs with PAs was TSKgel IC-Anion-SW (silica gel-based anion-exchange resin with 5 µm particle size and 0.03 mmol g-1 ion-exchange capacity, packed in a 4.6 mm i.d. × 50 mm PTFE column). Amino-silica gel was prepared as follows: 5 g of silica gel (Wakosil 5SIL, 5 µm particle size) was dispersed in 100 mL of dry toluene containing with 5 g of (3-aminopropyl)triethoxysilane. After the solution was mixed for 1 h at room temperature, the silica gel was filtered and washed repeatedly with acetone, methanol, and water; this stationary phase was dried under vacuum at 70 °C. A diethylenetriamine (dien) column was prepared as follows: the same silica gel (2 g) as used for the (10) Ohki, A.; Okamoto, J.; Naka, K.; Maeda, S. Chromatographia 1991, 32, 73-78. (11) Nishimura, M.; Hayashi, M.; Hayakawa, K.; Miyazaki, M. Anal. Sci. 1994, 10, 321-324. (12) Hosseini, M. W.; Lehn, J. M. J. Am. Chem. Soc. 1982, 104, 3525-3527. (13) Johansouz, H.; Jiang, Z. J. Am. Chem. Soc. 1989, 111, 1409-1413. (14) Iverson, B. L.; Thomas, R. E.; Kral, V.; Sessler, J. L. J. Am. Chem. Soc. 1994, 116, 2663-2664. (15) Whitlock, B. J.; Whitlock, H. W. J. Am. Chem. Soc. 1994, 116, 2301-2311. (16) Bayly, C. I.; Kollman, P. A. J. Am. Chem. Soc. 1994, 116, 697-703. 0003-2700/96/0368-1158$12.00/0

© 1996 American Chemical Society

potassium salts and used as received. Since PAs were formed from polyamines in sufficiently acidic solution, reagents used were not polyammonium ions but polyamines such as ethylenediamine [en(2+), where a charge under experimental conditions is given in parentheses], 1,3-propanediamine [pn(2+)] 1,4-butanediamine [bn(2+)], 1,6-hexanediamine [hn(2+)], 1,8-octanediamine [on(2+)], 1,10-decanediamine [dn(2+)], diethylenetriamine [dien(2+) and dien(3+)], triethylenetetramine [trien(4+)], tetraethylenepentamine [tetraen(5+)], pentaethylenehexamine [pentaen(6+)], N-(2-aminoethyl)-1,3-propanediamine [enpn(3+)], 3,3′-diaminodipropylamine [dipn(3+)], 1,2-propanediamine [Me-en(2+)], N,N,N′,N′-tetramethylethylenediamine [tetraMe-en(2+)], and N,N,N′,N′′,N′′-pentamethyldiethylenetriamine [pentaMe-dien(3+)]. These PAs were purchased from Aldrich [on(2+) and enpn(3+)], Nacalai Tesque [pentaen(6+)], and Wako Pure Chemicals (others) and used as received. Distilled, deionized water was used for the preparation of solutions. Ion-pair formation constants were determined by studying the dependence of the retention of ADSs on the concentration of a polyammonium ion in the mobile phase containing both 50 mM NaClO4 and 50 mM formic acid. The calculation of molecular sizes and electrostatic energies was carried out with a CAChe system on a Macintosh Quadra 650.

Figure 1. Structures and abbreviations of compounds used in the present investigation.

RESULTS AND DISCUSSION Ion-Pair Formation Constants between ADSs and PAs. Ion-pair formation constants between ADSs and PAs were determined by anion-exchange chromatography. To determine the constants, 1:1 stoichiometry was assumed: KIP

preparation of amino-silica gel was dispersed in 50 mL of dry toluene containing 2 g of (3-chloropropyl)trimethoxysilane. After the solution was mixed for 1 h at room temperature, chloropropylated silica gel was filtered and washed repeatedly with acetone, methanol, and water. After being dried, the silica gel was added to 50 mL of dioxane containing 10 g of dien. After 2 h of reflux, the stationary phase (dien-silica gel) was filtered, washed, and dried under vacuum at 70 °C. These stationary phases were packed in 4.6 mm i.d. × 50 mm PTFE columns in the usual manner and protonated by aqueous HClO4 solution before use. The amounts of introduced groups per column were 0.141 mmol for amino-silica gel and 0.0212 mmol (in full protonation, corresponding to 0.0637 mmol ion-exchange capacity) for dien-silica gel. These were indirectly determined by investigating the amounts of counterions bound by the cationic stationary phases. The capillary electrophoretic system was composed of a highvoltage power supply (Model HCZE 30P, Matsusada Precision Devices) and a UV detector (Model 870-CE, JASCO). A fusedsilica capillary (50 µm i.d., 375 µm o.d., and 60 cm long) was used for CE separation. The detection window was located at 15 cm from the ground end. Sample solution was introduced into the capillary by siphoning. Figure 1 shows the structures and abbreviations of compounds studied in the present paper. Anthraquinone-1,5-disulfonate (1,5AnDS), anthraquinone-1,8-disulfonate (1,8-AnDS), naphthalene1,5-disulfonate (1,5-NDS), naphthalene-2,6-disulfonate (2,6-NDS), and naphthalene-2,7-disulfonate (2,7-NDS) were obtained from Wako Pure Chemicals (1,5-AnDS and 2,7-NDS), Tokyo Kasei (1,8AnDS and 1,5-NDS), and Nacalai Tesque (2,6-NDS) as sodium or

ADS2- + PAn+ {\} ADS PAn-2

Anion-exchange chromatographic retention is affected by the formation of an ion-pair with PAn+ added in the mobile phase. Resulting ion-pairs were zero or positive charges are not retained on the resin. In this instance, the capacity factor (k′) of ADSs is represented by

k′ ) φ[ADS(s)]/{[ADS(m)] + [ADS PA(m)]} ) φ[ADS(s)]/{[ADS(m)] + CPAKIP[ADS (m)]} 1/k′ ) (1 + CPAKIP)/k′0

where CPA and k′0 denote the concentration of PAs added in the mobile phase and the capacity factor of a solute obtained with CPA ) 0, and s and m in parentheses represent the stationary and the mobile phases. Thus, we can chromatographically determine KIP by studying the dependence of k′ on CPA. Table 1 summarizes KIP values as well as standard deviations. Charges of the PAs were controlled by changing the pH of the mobile phase. To avoid large changes in ionic strength, ranges of CPA used for studies are different for the different charges of PAs; e.g. 0-16 mM for divalent PAs, 0-3 mM for trivalent PAs, 0-2 mM for trivalent or more highly charged PAs. Since k′ values are more sensitive to the changes in the concentrations of PAs of larger charges, narrow CPA ranges caused no experimental problems. Analytical Chemistry, Vol. 68, No. 7, April 1, 1996

1159

Table 1. Ion-Pair Formation Constants (KIP) between ADSs and PAs with Standard Deviations (σ) log KIP (σ)

en(2+) pn(2+) bn(2+) hn(2+) on(2+) dn(2+)a dien(2+) dien(3+) trien(4+) tetraen(5+) pentaen(6+) enpn(3+) dipn(3+) Me-en(2+) tetra-Me-en(2+) penta-Me-dien(3+) a

1,5-AnDS

1,8-AnDS

1,5-NDS

2,6-NDS

2,7-NDS

0.95 (0.08) 0.85 (0.07) 0.93 (0.10) 1.06 (0.02) 1.17 (0.03) 1.36 (0.04) 1.06 (0.09) 1.48 (0.06) 1.70 (0.04) 1.79 (0.05) 1.92 (0.03) 1.31 (0.04) 1.43 (0.07) 0.98 (0.02) 0.85 (0.04) 0.73 (0.27)

1.96 (0.02) 1.71 (0.01) 1.64 (0.04) 1.49 (0.01) 1.30 (0.04) b 1.91 (0.02) 2.85 (0.01) 3.32 (0.00) 3.57 (0.00) 3.77 (0.01) 2.55 (0.01) 2.32 (0.01) 1.92 (0.01) 1.63 (0.02) 1.71 (0.06)

0.78 (0.10) 0.80 (0.01) 0.98 (0.02) 1.19 (0.01) 1.23 (0.02) 1.30 (0.02) 1.09 (0.07) 1.38 (0.07) 1.58 (0.03) 1.75 (0.03) 1.92 (0.01) 1.17 (0.06) 1.48 (0.05) 0.88 (0.03) 1.00 (0.04) 1.29 (0.08)

0.70 (0.12) 0.73 (0.05) 0.92 (0.07) 1.18 (0.02) 1.26 (0.01) 1.41 (0.01) 1.09 (0.07) 1.31 (0.09) 1.50 (0.07) 1.62 (0.05) 1.79 (0.12) 0.99 (0.10) 1.37 (0.06) 0.86 (0.03) 0.89 (0.07) 1.18 (0.13)

0.79 (0.11) 0.79 (0.05) 0.98 (0.07) 1.21 (0.02) 1.29 (0.01) 1.42 (0.01) 1.14 (0.06) 1.40 (0.09) 1.62 (0.05) 1.75 (0.03) 1.91 (0.07) 1.19 (0.09) 1.43 (0.05) 0.92 (0.03) 0.97 (0.06) 1.26 (0.13)

20% methanol solution was used because of low solubility of dn in water. b Not determined.

Table 2. Lengths between Ionic Sites in ADSs and PAs distance/ Å 1,8-AnDS 1,5-AnDS 1,5-NDS 2,6-NDS 2,7-NDS b c d

5.46 8.36 6.94 8.60 7.97

distance/ Å en(t)a en(g)a pn(tt)b pn(g+g+)b pn(g+g-)b

3.77 2.98 4.91 3.73 3.03

distance/ Å dien(t-t)c dien(g-t)c dien(g+-g+c dien(g+-g-)c

7.45d 6.40d 5.29d 4.67d

a t and g in parentheses denote trans and gauche along C-C bond. Conformation along C-C-C bonds for pn is shown in parentheses. Conformation along two C-C bonds in dien is shown in parentheses. Distance between terminal ammonium ions.

Some interesting features involved in these data are summarized as follows: (1) KIP values between 1,8-AnDS and R,ωalkylenediammonium ions decrease with increasing number of methylene units between terminal ammonium ions, whereas corresponding values for other ADS increase; (2) for the oligomeric series of ethylenediammonium, KIP values increase with increasing number of repeating ethyleneammonium units for any ADS; and (3) this increase in KIP values for 1,8-AnDS is marked in comparison with those for other ADS. It is well-known that increasing hydrophobicity of the molecules involved in the reaction causes stable ion-pair formation; i.e., since the water structure is enhanced around hydrophobic ions, ion-pair formation allows a decrease in the surface area and results in an increase in entropy. Such phenomena have been reported in many instances.17,18 Table 2 lists the distances between sulfur atoms in ADS molecules and those between nitrogen atoms in PAs. According to the concept of supramolecular chemistry (molecular ruler), the match of span between interacting sites in a host molecule with that in a guest molecule is essential for stable complexation.19 According to this concept, the match of the distance between sulfonate ions in an ADS with that between nitrogen atoms in a polyammonium ion is important for 1:1 ion-pair formation in the (17) Barron, R. E.; Fritz, J. S. J. Chromatogr. 1984, 284, 13-25. (18) Melander, W. R.; Horvath, C. In Ion-Pair Chromatography: Theory and Biological and Pharmaceutical Application; Hearn, M. T. W., Ed.; Marcel Dekker: New York, 1985; Chapter 2. (19) Pirkle, W. H.; Readnour, R. S. Anal. Chem. 1991, 63, 16-20.

1160 Analytical Chemistry, Vol. 68, No. 7, April 1, 1996

present instance. However, the rather flexible structures of polyammonium ion molecules complicate the situation, and we must take their conformational changes into account. Distances between sulfonic acids in ADSs (S-S distance) except for 1,8-AnDS range from 6.94 to 8.6 Å, and those between ammonium ions in en(2+) are 3.77 Å for a trans conformer (C-C bond) and 2.98 Å for a gauche conformer. It seems that the distance between ammonium ions in en(2+) is too short to allow stable interaction with sulfonate groups. It was therefore assumed that interaction of terminal ammonium sites in dien(3+) with ADSs results in formation of a stable ion-pair. As shown in Table 2, the distances between terminal ammonium sites in dien(3+) are 7.45, 6.40, 5.29, and 4.67 Å for t-t, g-t, g+-g+ (or g--g-), and g+-g- (or g--g+) around C-C bonds (for simplicity, trans conformation was assumed for C-N bonds). If dien(3+) adopts complete trans conformation, the size of the molecule fits to the S-S distances of 1,5-AnDS and 1,5-, 2,6-, and 2,7-NDS. According to this consideration, increases in KIP with increasing number of ethyleneammonium units should be principally explained by an entropy effect resulting from statistical increases in interacting sites. Figure 2 shows changes in KIP with the number of ethyleneammonium units, together with a corresponding increase in KIP calculated by taking only entropy effects into account. The calculated results almost agree with experimental values, except for 1,8-AnDS. The above consideration could not explain the results for 1,8AnDS. The determination of thermodynamic parameters was difficult because of almost athermal equilibria. Many ion-pair formation equilibria in water are also athermal or endothermic, suggesting that the electrostatic energy gain is compensated by the dehydration enthalpy of ions, and eventually the entropic gain accompanying the release of solvated water molecules thermodynamically dominates the ion-pair formation process.20 Since we do not quantitatively determine the degree of the desolvation upon the ion-pair formation, complete dehydration was assumed, and the contribution of electrostatic energies was calculated for the ion-pair formation of 1,8-AnDS with en(2+) or pn(2+). (20) Gill, J. B. In Chemistry of Nonaqueous Solutions: Current Progress; Mamantov, G., Popov, A. I., Eds.; VCH: New York, 1994; Chapter 2.

Figure 2. Changes in KIP values with the number of repeating ethyleneammonium units in oligoethyleneammonium ions. The dashed line indicates a change in KIP values predicted by statistical increase in the number of possible interactive sites, assuming the interaction of terminal primary ammonium ions in dien(3+).

Figure 3. Electrostatic energies of possible interaction between 1,8AnDS and en(2+) or pn(2+) calculated by molecular mechanics.

We used the MM2 force field to estimate electrostatic energies. Final energy obtained with MM2 calculation involves energies of bond stretch, bond angle, dihedral angle, improper torsion, van der Waals, electrostatics, and hydrogen bonding. Since energy terms other than electrostatic energy are not necessary for the present purpose, calculation was carried out for the fixed conformation of a polyammonium ion, and the total energy was minimized in terms of the relative position of the PA and 1,8AnDS. Results are illustrated in Figure 3. For en(2+), the electrostatic interaction energy of a gauche conformer (Figure 3a) was more negative than that of a trans conformer (Figure 3b). In contrast, for pn(2+), the energy for a t-t conformer (Figure

3d) is more negative than that for a g-g conformer (Figure 3c). As shown above, the match of the distances between ionic sites of the molecules involved in an ion-pair formation equilibrium is an important factor for stable ion-pair formation. According to this concept, the distance between terminal ammonium ions in en(2+) is too short to allow interactions with both sulfonate sites even when en(2+) adopts a trans conformation. In contrast, the molecular size of the t-t conformer of pn(2+) fits the S-S distance of 1,8-AnDS. However, molecular mechanics calculation gave the most negative electrostatic energy to the interaction of the gauche conformer of en(2+), as shown in Figure 3, because a terminal ammonium ion of en(2+) is trapped in a pocket between two sulfonates in 1,8-AnDS. The data listed in Table 1 imply the importance of terminal primary ammonium ions in the ion-pair formation of 1,8-AnDS. Log KIP values of 1,8-AnDS with en(2+), Me-en(2+), and tetraMeen(2+) are 1.96, 1.92, and 1.62, and those with dien(3+) and pentaMe-dien(3+) are 2.85 and 1.72; the difference between en(2+) and tetraMe-en is 0.34 ( 0.03, and that between dien(3+) and pentaMe-dien(3+) is 1.13 ( 0.06. In contrast, decreases in corresponding differences for other ADS are much smaller (e.g., for 2,7-NDS, 0.13 ( 0.11 and 0.14 ( 0.16). These results also support the interaction shown in Figure 3a: a bulky ammonium ion (e.g., N-methyl-substituted) is unfavorably trapped in the pocket of 1,8-AnDS because of steric hindrance. If one terminal ammonium ion of an R,ω-alkylenediammonium ion is trapped in the pocket of 1,8-AnDS, then the other terminal ammonium site should be placed outside the pocket and located farther from sulfonate sites as an intermediate hydrocarbon chain becomes longer. This causes weaker electrostatic attraction and eventually lowers the KIP. Modification of Anion-Exchange Chromatographic Selectivity. Use of the ion-pair formation mentioned above allows the modification and enhancement of the anion-exchange chromatographic selectivity of ADS. The elution order of ADSs from an IC-Anion-SW column with ClO4- mobile phase was 1,5-AnDS < 1,8-AnDS < 2,6-NDS < 1,5-NDS < 2,7-NDS, as shown in Figure 4A. Though the details have not been elucidated, it appears that the retention is large when two sulfonate sites are located on the same side of a molecule. This selectivity cannot be changed in the absence of ion-pair formation in the mobile phase. When an appropriate PA is added in the mobile phase, 1,8-AnDS forms much more stable ions-pairs with the PA than do the others, and its retention time is reduced. Figure 4B shows the inversion in the elution order of 1,8-AnDS and 1,5-AnDS upon the addition of dien(3+) in the mobile phase. Unfortunately, the similarity of the ion-pair formation of naphthalene disulfonates did not improve the separation of these compounds. Stationary Phase Selectively Retaining 1,8-AnDS. High stability constants of ion-pair formation between 1,8-AnDS and PAs bearing the ethylenediammonium structures lead to the idea that such interaction can be used for the design of selective separation. As already shown in Figure 4, the retention of 1,8-AnDS on anionexchange resins having tetraalkylammonium ions as ion-exchange sites is weaker than that of NDS. Although ion-pair formation in the mobile phase reduced the retention, similar reaction in the stationary phase should increase the retention; 1,8-AnDS can be retained more strongly than NDS. In the present instance, silica gel modified by dien was synthesized and used to check the retention behaviors. IonAnalytical Chemistry, Vol. 68, No. 7, April 1, 1996

1161

Figure 4. Inverse in the separation selectivity by adding PAs in the mobile phase. Stationary phase, TSKgel IC-Anion-SW; mobile phase, (A) 0.05 M NaClO4 (pH ) 2.8), (B) 0.05 M NaClO4 + 12 mM dien (pH ) 2.8); concentration of ADS, 5 × 10-5 M; detection, 250 nm. Figure 6. Electrophoretic separation of ADS. Electrophoretic conditions: voltage, 10 kV; current, 10 µA; anodic detection for A, B, and C and cathodic detection for D. Solution: (A) 0.02 M NaClO4 + 0.02 M formic acid (pH ) 2.8); (B) A + 8 mM dien; (C) and (D) A + 2 mM pentaen. Peak identification: ref, reference (NO3-); 1, 2,6NDS; 2, 2,7-NDS; 3, 1,5-NDS; and 4, 1,8-AnDS. Concentration of ADS was 0.5 mM. Detection was at 250 nm.

Figure 5. Selective retention of 1,8-AnDS with dien-bonded stationary phase. Stationary phase, (upper) dien-bonded silica gel, (lower) NH2-silica gel; mobile phases, 0.1 M KH2PO4 + 10 mM NaOH (pH ) 5.8) for NH2-silica gel and 0.02 M KH2PO4 + 2 mM NaOH (pH ) 5.8) for dien-silica gel; concentration of ADS, 5 × 10-5 M; detection, 250 nm.

exchange selectivity is changed by the structure of ion-exchange sites, even in the absence of specific ion-pair formation. In particular, there is a large difference in selectivity between primary ammonium and tertiary ammonium ion-exchange sites.21 To minimize such effects and facilitate the comparison of results, the separation selectivity with NH3+-silica gel was tested. As shown in Figure 5, the elution order with NH3+-silica gel is 2,6-NDS < 1,5-NDS < 1,8-AnDS < 2,7-NDS. This order is changed to 2,6NDS < 1,5-NDS (not shown for clarity) < 2,7-NDS , 1,8-AnDS with dien-silica gel. Relatively basic mobile phases (pH ) 5.8) are used to compare retention times obtained with these two stationary phases. In such (21) Okada, T. Bunseki Kagaku 1995, 44, 579-601 and references therein.

1162 Analytical Chemistry, Vol. 68, No. 7, April 1, 1996

media, dien is not completely protonated and is in the form dien(2+) if the acid dissociation constants of dien in water are applicable to the present case. Moreover, mobile phase concentrations are not identical because of different anion-exchange capacity. Despite these facts, the ability of dien stationary phase to selectively retain 1,8-AnDS is remarkable. Though comparison of the retention times was impossible, the dien stationary phase more selectively retained 1,8-AnDS with SO42- mobile phase (pH ) 2.5) than under the above condition; a ratio of k′ for 1,8-AnDS to that for 2,7-NDS was 21, much larger than the values described below. As predicted from similar ion-pair formation constants of other ADSs with dien(2+), a use of the dien(2+)-type stationary phase did not improve the separation of these solutes. The ratios of k′ for 1,8-AnDS to k′ for 2,7-NDS are 3.6 for dien(2+)-silica gel and 0.81 for NH3+-silica, implying specific ion-pair formation in the stationary phase. Capillary Electrophoretic Separation of ADSs. The separation of compounds having almost the same size and the same charge is difficult, even with such an effective separation means as capillary electrophoresis (CE), as shown in Figure 6A. Terabe and Isemura22 reported that use of a polymeric pseudo-stationary phase allowed the separation of naphthalenedisulfonates having identical electrophoretic mobility. The present results also imply that the use of PAs improves the separation of ADSs, albeit the difference in KIP is very small. CE was performed under an acidic condition (pH ) 2.8), and detection was carried out at the anodic end unless otherwise stated. Since electroosmotic flow (EOF) is small under acidic (22) Terabe, S.; Isemura, T. Anal. Chem. 1990, 62, 650-652.

conditions, NO3- was added in samples to check the effect of EOF. It is interesting to note that the peak for 1,8-AnDS is not detected by anodic detection when dien(3+) is added to the solution because this compound forms a very stable, positively charged ion-pair with dien(3+), which is not detected by a detector placed at the anodic end. However, as shown in Figure 6B, a use of the solution containing dien(3+) permitted the separation of 2,6-NDS from 1,5NDS and 2,7-NDS. The addition of trien(4+) and tetraen(5+) gave similar results. However, complete separation was made possible by the use of pentaen(6+). Figure 6C illustrates an example of the separation. As shown in Table 1, the difference in KIP values between 1,5-NDS and 2,7-NDS is very small, even when pentaen(6+) is used as an ion-pairing reagent. Therefore, the separation between 1,5-NDS and 2,7-NDS is not caused by a subtle difference in KIP. There are two possible reasons that the separation was improved by adding pentaen(6+): one is an enlarged difference in the mobility of an ion-pair, and the other is reduced overall mobility due to the large positive charges (tetravalent) of an ion-pair of pentaen(6+). As we could not measure the rate of very small EOF, reliable evaluation of the mobilities of ion-pairs was not possible. In contrast, the reduced (23) Vindevogel, J.; Sandra, P. Introduction to Micellar Electrokinetic Chromatography; Huthig: Heidelberg, 1992; pp 31-34.

overall mobility does improve the resolution, as is well-known.23 However, the addition of pentaen(6+) to the running electrolyte results in the obvious enhancement of the selectivity that cannot be explained by the reduced overall mobility alone, suggesting the importance of the mobility of a resulting ion-pair. Another electropherogram (Figure 6D) shows a peak of 1,8AnDS obtained by cathodic detection under the same condition as was used for Figure 6C. Strong ion-pair formation of 1,8-AnDS results in the overall cationic charge and the movement to the cathode. Despite the effectiveness of the addition of PA to the running electrolyte, 1,5-AnDS was still coeluted with 2,7-NDS, and simultaneous separation of these five ADS was not possible. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

Received for review November 2, 1995. Accepted January 15, 1996.X AC951096W X

Abstract published in Advance ACS Abstracts, February 15, 1996.

Analytical Chemistry, Vol. 68, No. 7, April 1, 1996

1163