Anal. Chem. 1998, 70, 2516-2522
Anion Selectivity of a Sapphyrin-Modified Silica Gel HPLC Support Jonathan L. Sessler,* Vladimı´r Kra´l, John W. Genge, Richard E. Thomas, and Brent L. Iverson*
Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712
A sapphyrin-modified silica gel support for use in highperformance liquid chromatography was prepared by attaching a sapphyrin monocarboxylic acid to aminopropyl silica gel through an amide bond. The anion retention characteristics of this modified silica gel were tested by exploring the extent to which a specific anion in the mobile phase would act to affect the rate at which AMP was eluted from an HPLC column containing this functionalized stationary phase. In general, it was found that phosphate and arsenate anions were more effective as eluents than carboxylic acids and halides, a result that was interpreted in terms of these former species binding better to sapphyrin (and hence being more effective in terms of displacing AMP) than other anions tested. Support for the contention that phosphate anions will bind to sapphyrin subunits covalently tethered to the silica gel came from solid state 31P NMR spectroscopic analyses. These revealed that the 31P nucleus undergoes a 5 ppm upfield shift, relative to control, when allowed to interact with the sapphyrin-containing support.
The benefits associated with attaching supramolecular structures to solid supports for the purposes of cation separation have been recognized for more than 20 years. In early work, Cram et al. attached chiral naphthal crown ethers to polystyrene resins and used the resulting supports to achieve the separation of certain D- and L-amino acids including phenylglycine perchlorate.1 Nearly contemporaneously, Blasius and co-workers attached crown ether monomers and dimers to silica gel and began studying the way in which the resulting systems could be used to separate monovalent cations.2 These workers found that silica gels modified with dimeric crown ethers could be used to separate alkali cations in accord with the following retention order: Li+ < Na+ < Rb+ < K+ < Cs+. Subsequent work from the groups of Izatt and Bradshaw then served to show, inter alia, that silica gel bound azacrown ethers possessed nearly the same cation-binding affinities as did the analogous unbound azacrown ethers.3-5 (1) Sogah, G. D. Y.; Cram, D. J. J. Am. Chem. Soc. 1976, 98, 3038-3041. (2) (a) Blasius, E.; Janzen, K.-P.; Adrian, W.; Klein, W.; Klotz, H.; Luxemburger, H.; Mernke, E.; Nguyen, V. B.; Nguyen-Tien, T.; Rausch, R.; Stockemer, J.; Tossaint, A. Talanta 1980, 27, 127. (b) Blasius, E.; Janzen, K.-P.; Keller, M.; Lander, H.; Nguyen-Tien, T.; Scholten, G. Talanta 1980, 27, 107. (c) Blasius, E.; Janzen, K.-P.; Adrian, W.; Klautke, G.; Lorscheider, R.; Maurer, P.-G.; Nguyen, V. B.; Nguyen-Tien, T.; Scholten, G.; Stockemer, J. Fresnius’ Z. Anal. Chem. 1977, 284, 337.
2516 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
In contradistinction to what is true for cations, the use of recognition elements supported on solid matrixes is a rather undeveloped approach when applied to the problem of anion separation. One of the earliest examples of anion chromatography utilizing conjugated macrocyclic chemistry was reported by Kokufuta and co-workers and involved the use of oxochloromolybdenum(V) tetraphenylporphyrin encased in a polystyrene matrix.6 This support displayed a selectivity for H2PO4- that proved superior to conventional quaternary ammonium-based anion exchange supports. Kibbey and Meyerhoff have also contributed substantially to the field of supramolecular-based anion separations.7 These workers have done this in part by attaching tetraphenylporphyrin (TPP) to a trimethylsilyl-protected silica gel via an amide linkage.7a The resulting support was then metalated to give the corresponding tin(IV) or indium(III) derivatives (SnTPP-silica gel and In-TPP-silica gel, respectively). Both the indium(III) and the tin(IV) tetraphenylporphyrin-substituted silica gels could be used to separate inorganic anions and purify certain aromatic anions, including the benzoate anion. The Sn-TPPsilica gel was also found to display species selectivity within a given class of generalized substrates.8 For example, it was found that individual anionic constituents within a mixture of aromatic sulfonates could be separated by HPLC using this support. Surprisingly, despite this success, these supports were not (apparently) used to effect the separation of such biologically active molecules as nucleotides, amino acids, or oligonucleotides. We recently became interested in using nonporphyrinic polypyrrole macrocycles to generate anion-separating chromatography media.9,10 The bulk of this work has involved the use of sapphyrin (3) Bradshaw, J. S.; Krakowiak, K. E.; Tarbet, B. J.; Bruening, R. L.; Biernat, J. F.; Bochenska, M.; Izatt, R. M.; Christensen, J. J. Pure Appl. Chem. 1989, 61, 1619-1624. (4) Bradshaw, J. S.; Bruening, R. L.; Krakowiak, K. E.; Tarbet, B. J.; Bruening, M. L.; Izatt, R. M.; Christensen, J. J. J. Chem. Soc., Chem. Commun. 1988, 812-814. (5) Izatt, R. M.; Bruening, R. L.; Tarbet, B. J.; Griffin, D.; Bruening, M. L.; Krakowiak, K. E.; Bradshaw, J. S. Pure Appl. Chem. 1990, 62, 1115-1118. (6) Kokufuta, E.; Sodeyama, T.; Takeda, S. Polym. Bull. 1986, 15, 479-484. (7) (a) Kibbey, C. E.; Meyerhoff, M. E. Anal. Chem. 1993, 65, 2189-2196. (b) Kliza, D. M.; Meyerhoff, M. E. Electroanalysis 1992, 4, 841-849. (c) Xiao, J.; Savina, M. R.; Martin, G. B.; Francis, A. H.; Meyerhoff, M. E. J. Am. Chem. Soc. 1994, 116, 9341-9342. (8) Meyerhoff’s TPP-based silica gel may also be used to separate neutral aromatic species such as naphthalene, anthracene, imidazole, and pyridine. In this case, π-π interactions between TPP and the neutral substrates probably play a critical role in mediating the separation process (see ref 7a). (9) (a) Iverson, B. L.; Thomas, R. E.; Kra´l, V.; Sessler, J. L. J. Am. Chem. Soc. 1994, 116, 2663-2664. (b) Sessler, J. L.; Genge, J. W.; Kra´l, V.; Iverson, B. L. Supramol. Chem. 1996, 8, 45-52. S0003-2700(97)01214-6 CCC: $15.00
© 1998 American Chemical Society Published on Web 05/15/1998
as the key anion-binding recognition unit.9a Sapphyrin is a pentapyrrolic, 22-π-electron, expanded porphyrin reported first by Woodward.11,12 It has been shown to be monoprotonated at physiological pH13 and to bind certain anionic species well, including phosphates.13-15 A variety of spectroscopic studies,14,16,17 as well as X-ray crystallographic analyses,18,19 have served to show that anionic and phosphorylated substrates are bound to the protonated macrocyclic sapphyrin core via close contacts involving the pyrrolic hydrogens and the phosphate oxyanion. Consistent with these suppositions were the findings that sapphyrin, once attached to a silica gel solid support, could be used to effect the HPLC-based separation of mono-, di-, and triphosphate nucleotides, as well as short oligonucleotides, under conditions of isocratic elution.9a Other oxyanions, such as phenylarsenate, phenyl hydrogen phosphate, and benzenesulfonate, could also be separated using these sapphyrin supports. However, the selectivities observed were seemingly nonobvious. In fact, the observed retention times were found to correlate best not with conjugate acid pKa but with heteroatom-to-oxygen bond length of the presumably bound anionic substrate. To test further the validity of the latter conclusion and to learn more about the anion retention characteristics of protonated sapphyrins, we have now carried out a detailed HPLC-based anion competition analysis that involves the use of a sapphyrin-bearing silica gel column. This analysis, which was made by looking at the relative rates of from-column AMP displacement (i.e., elution times), serves to confirm that sapphyrin-based supports do indeed display inherent anion selectivities. Control experiments, also reported here, reveal that these selectivities are not mirrored in analogous porphyrin-based systems. EXPERIMENTAL SECTION General Methods. 31P NMR spectra were recorded on a Nicolet NT-360 (81 MHz) spectrometer with phosphoric acid being used as an external reference. 1H NMR spectra were recorded on a General Electric QE 300 (300 MHz) spectrometer. All modified silica gel columns were packed commercially by Alltech Associates, Inc. (Deerfield, IL), in 4.6 mm × 100 mm stainless steel HPLC columns. The HPLC system used in this work consisted of a Varian (Walnut Creek, CA) 9002 solvent delivery system, a Varian 9050 variable-wavelength UV-visible detector, (10) Sessler, J. L.; Gale, P. A.; Genge, J. W. Chem.sEur. J., in press. (11) Bauer, V. J.; Clive, D. L. J.; Dolphin, D.; Paine, J. B., III; Harris, F. L.; King, M. M.; Loder, J.; Wang, S. W. C.; Woodward, R. B. J. Am. Chem. Soc. 1983, 105, 6429-6436. (12) Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc., Perkin Trans. 1 1972, 2111-2116. (13) Shionoya, M.; Furuta, H.; Lynch, V.; Harriman, A.; Sessler, J. L. J. Am. Chem. Soc. 1992, 114, 5714-5722. (14) Sessler, J. L.; Cyr, M.; Furuta, H.; Kra´l, V.; Mody, T.; Morishima, T.; Shionoya, M.; Weghorn, S. Pure Appl. Chem. 1993, 65, 393-398. (15) Sessler, J. L.; Cyr, M. J.; Lynch, V.; McGhee, E.; Ibers, J. A. J. Am. Chem. Soc. 1990, 112, 2810-2813. (16) Sessler, J. L.; Kra´l, V. Tetrahedron 1995, 51, 539-554. (17) (a) Iverson, B. L.; Shreder, K.; Kra´l, V.; Sansom, P.; Lynch, V.; Sessler, J. L. J. Am. Chem. Soc. 1996, 118, 1608-1616. (b) Kra´l, V.; Furuta, H.; Shreder, K.; Lynch, V.; Sessler, J. L. J. Am. Chem. Soc. 1996, 118, 1595-1607. (c) Iverson, B. L.; Shreder, K.; Kra´l, V.; Smith, D. A.; Smith, J.; Sessler, J. L. Pure Appl. Chem. 1994, 66, 845-850. (18) Unpublished result. (19) (a) Iverson, B. L.; Shreder, K.; Kra´l, V.; Sessler, J. L. J. Am. Chem. Soc. 1993, 115, 11022-11023. (b) Sessler, J. L.; Brucker, E. A.; Lynch, V.; Choe, M.; Sorey, S.; Vogel, E. Chem.sEur. J. 1996, 2, 1527-1532.
a Varian Star chromatography workstationsversion 3, and a Rheodyne (Cotati, CA) model 7125 sample valve with a 20-µL loop. Elemental analyses were determined by Atlantic Microlab (Atlanta, GA). Binding Studies. Association constants were determined in solution using 1H NMR spectroscopic titrations carried out in methanol-d4. The concentration of the phenyl-substituted acid was held constant at a chosen value ranging from 0.3 to 3 mM, and the change in the chemical shift of its meta proton was recorded as a function of increasing free-base 3,12,13,22-tetraethyl-2,7,18,23-tetramethyl-8,17-bis(hydroxypropyl)sapphyrin concentration. Data reduction was performed using standard methods.20 Apparent association constants were determined using iterative, curvefitting procedures wherein an initial guess was entered and variables, such as concentration and chemical shift, were calculated using EQNMR.21 It is known from previous studies17a that the protonated forms of sapphyrin are capable of forming strong 1:1 sapphyrin-anion complexes and much weaker 1:2 sapphyrin-anion complexes. For this reason, anion (or more precisely phenyl-substituted conjugate acid) concentrations were kept below 3 mM. In line with what was true in the case of earlier, but analogous, 1H NMR spectroscopic titration studies,17b it was generally assumed that at these concentrations 1:1 binding constituted the predominant mode of interaction. Further, since the nonprotonated (free-base) form of sapphyrin was being titrated with the acid form of the anionic substrates, it was assumed that the binding observed involved interactions between the anion in question and the monoprotonated form of sapphyrin. The Ka values determined in this study thus correspond to K11 processes reported previously.17b Solid State 31Phosphorus NMR Spectroscopic Studies. Samples for the solid state 31P NMR spectral studies were prepared in one of two ways, as necessity dictated. The first method involved stirring 3 × 10-4 mol of AMP/g of sapphyrin-functionalized or trimethylsilyl-protected (“capped”) 3-aminopropyl silica gel in water and then removing the solvent in vacuo. The second approach involved using a 1:1 ratio of monoprotonated, molecular (i.e., free) 3,12,13,22-tetraethyl-2,7,18,23-tetramethyl-8,17-bis(hydroxypropyl)sapphyrin and AMP. In this case, the two species were both dissolved in water and stirred together. The water was then removed and the resulting mixture combined with sapphyrinfree (but other wise “capped”) silica gel in a ratio equal to that described above. The spectra themselves were recorded using the following conditions: phosphoric acid as external standard; sweep width, 50 kHz; spin rate, 5000 Hz; and a line broadening setting of 5 Hz. In general, 512 scans were recorded while magic angle spin coupling (MAS) was used, and a pulse delay of 10 s was employed. “Monohook” Sapphyrin Synthesis. The sapphyrin monocarboxylic acid derivative, 3,8,17,22-tetraethyl-12-(carboxyethyl)2,7,13,18,23-pentamethylsapphyrin (1), was prepared in accord with a previously published procedure.22 Preparation of Sapphyrin-Functionalized Silica Gel. 3-Aminopropyl silica gel (4 g) (Phase Separations) was suspended in a (20) Connors, K. A. Binding Constants. The Measurement of Molecular Complex Stability; J. Wiley: New York, 1987. (21) Hynes, M. J. J. Chem. Soc., Dalton Trans. 1993, 311-312. (22) (a) Kra´l, V.; Sessler, J. L. Tetrahedron 1995, 51, 539-554. (b) Kra´l, V.; Sessler, J. L.; Furuta, H. J. Am. Chem. Soc. 1992, 114, 8704-8705.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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solution of dry dichloromethane (100 mL), dry pyridine (2.0 mL, 24.72 mmol), and 4-(dimethylamino)pyridine (40 mg, 0.312 mmol). The monohook sapphyrin carboxylic acid, 1 (100 mg, 0.16 mmol), was likewise dissolved in dry dichloromethane (100 mL) and activated with N,N ′-diisopropylcarbodiimide (0.504 g, 4 mmol) and 1-hydroxybenzotriazole (20 mg, 0.148 mmol) at 0 °C for 40 min. The latter solution was then slowly added to the 3-aminopropyl silica gel slurry referred to above. The resulting reaction mixture was stirred at room temperature for 5 days. At this time, the silica gel was filtered off and washed with dichloromethane (120 mL), methanol (200 mL), water (200 mL), methanol (200 mL), and dichloromethane (200 mL). It was then dried under high vacuum for 2 days to give an isolated yield of 4.09 g. The product so obtained gave a microanalysis (C 5.00%, H 0.87%, N 0.47%) that could be compared with that of the unprotected aminopropyl silica gel (C 3.52, H 0.84%, N 0.47%). The free silanol groups were protected by suspending this crude derivatized sapphyrin silica gel product in 150 mL of dry dichloromethane and adding both dry pyridine (10 mL) and (trimethylsilyl)imidazole (5 g). The resulting suspension was then allowed to stir for 3 days. Workup of this newly silated silica gel mirrored the method used in the case of the nonsilated sapphyrin silica gel (described above). After isolation, the free amino groups were further protected by suspending the silated sapphyrin silica gel in dry dichloromethane (200 mL), adding the suspension, along with dry pyridine (15 mL) and acetyl chloride (3.5 mL), to a 250 mL round-bottom flask, and stirring the mixture for 3 days. The isolation and workup procedure then paralleled that used to obtain the original non-acetyl/non-silyl-protected sapphyrin-substituted silica gel described above. Monohook Porphyrin Synthesis. The porphyrin monocarboxylic acid derivative, 2-((methoxycarbonyl)ethyl)-8,12,13,17tetraethyl-3,7,18-trimethylporphyrin, was prepared in accord with a previously published procedure.23 Preparation of Porphyrin-Functionalized Silica Gel. This material was prepared from 3-aminopropyl silica gel (3 g) (Phase Separations) and the monohook porphyrin carboxylic acid (90 mg, 0.17 mmol) using a procedure identical to that described above (but with all reagents and solvents scaled to a three-fourths molar scale). The isolated yield of the crude porphyrin silica gel, whose microanalysis (C 4.20%, H 0.86%, N 0.46%) revealed a higher carbon percentage than that of the unprotected aminopropyl silica gel control (C 3.52%, H 0.84%, N 0.47%), was 3.06 g. The free silanol groups and amino groups were also “capped” using the same methodology as used in the sapphyrin case (vide supra). Workup and isolation, effected in a manner similar to that described for the original sapphyrin-substituted silica gel, provided a lavender silica gel product. Characterization of the Functionalized Silica Gels. The surface concentration of bonded species on the two silica gels was calculated according to the general equation proposed by Unger.24 Specifically, the extent of sapphyrin coverage on the aminopropyl silica was determined by recording the increase in carbon content prior to protection with the “capping” silating and acylating agents (cf. Table 1). The difference in carbon content (23) Sessler, J. L.; Genge, J. W.; Sansom, P. I.; Urbach, A. Synlett 1996, 187188. (24) Unger, K. K.; Becker, N.; Roumeliotis, P. J. Chromatogr. 1976, 125, 115127.
2518 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
Table 1. Elemental Analysis and Surface Coverage of Bonded Silica Gels bonded silica gel aminopropyl sapphyrin-functionalized porphyrin-functionalized
pore size surface area coverage (Å) (m2/g) % C (µmol/m2) 60 60 60
480 480 480
3.5 5.0 4.2
2.13 0.0685 0.0563
is 1.5% and corresponds to a sapphyrin coverage of 0.0685 µmol/ m2. The chromatographic efficiency of the sapphyrin-substituted silica gel column was determined from the HPLC elution of toluene with a mobile phase consisting of 50% methanol/50% water (v/v). The column efficiency (N) was calculated from the retention time of the toluene peak (tr) and its peak width at baseline (tw) as follows:
N ) 16(tr /tw)2 The sapphyrin-substituted silica gel column had an elution time and peak width over an average of four different runs (n ) 4) corresponding to an efficiency of N ) 4320 theoretical plates.7a Elution of AMP from the Macrocycle-Substituted Silica Gel Columns Using Various Anionic Buffers. A 10 µL injection of a 5 mM solution of AMP was eluted using the following conditions: mobile phase 160 mM aqueous anionic buffer, pH ) 6, 7, or 8 (adjusted with acetic acid or NaOH(aq)); flow rate 0.20 mL/min; column temperature 25 °C; UV detection at 262 nm (0.100 absorbance unit full scale (AUFS)). Elution of Anisic Acid, Benzoic Acid, and 4-Nitrobenzoic Acid from the Sapphyrin Silica Gel Column. A 10 µL injection of a 1 mM solution of anisic acid, benzoic acid, and 4-nitrobenzoic acid (1:1:1 molar ratio) was eluted using the following conditions: mobile phase 20 mM aqueous acetate solution, pH ) 7 (adjusted with acetic acid or NaOH(aq)); flow rate 0.20 mL/min; column temperature 25 °C; UV detection at 262 nm (0.100 AUFS). Elution of Sulfanilic Acid, Benzenesulfonic Acid, and 4-Nitrobenzenesulfonic Acid from the Sapphyrin Silica Gel Column. A 10 µL injection of a 1 mM solution of sulfanilic acid, benzenesulfonic acid, and 4-nitrobenzenesulfonic acid (1:1:1 molar ratio) was eluted using conditions identical to those described above with the notable exception that sulfate, rather than acetate, was used as the eluting anion. RESULTS AND DISCUSSION Column Preparation and Characterization. The reaction schemes shown in Figure 1 illustrate the methods used to couple the monohook sapphyrin carboxylic acid 1 to the aminopropyl silica gel. The reaction involves forming an amide bond between the carboxylic acid functionality and the amine group of the silica gel support. Scheme 1, shown in Figure 1, illustrates the method of coupling we used previously.9a It involves the generation of a sapphyrin acid chloride and the subsequent reaction with aminopropyl silica gel, a process that gives rise to the requisite covalent amide bond. Scheme 2, shown in this same figure, illustrates an improved coupling method. Here, diisopropyldiimide is reacted with the sapphyrin carboxylic acid to form an activated ester. This latter species then reacts with the amine group of the aminopropyl
Figure 1. Synthetic approaches to the sapphyrin-modified silica gels discussed in this report.
silica gel to form a linking amide bond identical to that produced from the acid chloride. An advantage of the newer carbodiimide-based method is that the active coupling species is formed in situ, which allows the carbodiimide to be used in excess to ensure a nearly complete coupling efficiency. Indeed, using this method, coupling yields of the carboxylic acid to the amine in excess of 95% were routinely recorded. By contrast, the acid chloride approach gave coupling yields on the order of 60%. AMP Elution Studies (Sapphyrin Column). This study has its genesis in the fact that our initial efforts to ascertain the anion selectivity of the sapphyrin-modified silica gel were hampered by what appeared to be an overly high anion affinity.9a Indeed, in our previous work with sapphyrin-based HPLC supports, we found that high buffer concentrations were needed to effect anion elution. This complicated the use of a conductivity detector and made it difficult to record directly the retention times of such classic anions as, e.g. chloride, bromide, and iodide. To circumvent this problem, an HPLC competition experiment was devised. Specifically, we chose to try eluting 5′-adenosine monophosphate (AMP) with buffered solutions of either pH 6, 7, or 8 that contained known, equivalent concentrations of various added anions (cf. Table 2). Here, the assumption was made that the strength of binding of the anion in question (to the sapphyrin silica gel) would be inversely proportional to the time needed to elute the AMP
Table 2. Times for AMP Elution from the Sapphyrin-Functionalized Silica Gel Using Different Anionic Mobile Phases retention time (min)
eluting anion (160 mM)
pH ) 6.0
pH ) 7.0
pH ) 8.0
arsenateb phosphateb sulfateb nitrateb trifluoroacetateb trichloroacetateb dichloroacetateb chloroacetateb acetateb chlorideb bromideb iodideb
6.3 ((0.05) 7.3 ((0.08) 12.9 ((0.07) 13.0 ((0.1) 18.5 ((0.3) 18.2 ((0.5) 18.3 ((0.4) 18.4 ((0.3) 19.0 ((0.2) 12.3 ((0.2) 13.0 ((0.3) 15.5 ((0.4)
5.3 ((0.2) 5.4 ((0.1) 10.3 ((0.1) 9.3 ((0.2) 18.8 ((0.8) 16.9 ((0.1) 17.1 ((0.4) 17.3 ((0.5) 21.0 ((0.9) 10.5 ((0.6) 10.9 ((0.5) 12.3 ((0.5)
4.4 ((0.1) 4.4 ((0.1) 8.0 ((0.2) 8.2 ((0.1) 11.0 ((0.1) 11.2 ((0.1) 11.1 ((0.2) 11.2 ((0.2) 11.0 ((0.1) 9.5 ((0.2) 10.0 ((0.3) 11.2 ((0.4)
bond lengtha (Å) 1.69 1.56 1.49 1.28 1.30 1.29 1.28 1.27 1.26
a Pearson, W. B. Structure Reports; D. Reidel Publishing Co.: Boston, MA, 1981. b pKa’s of the conjugate acid forms of the anions contained in the mobile phase are as follows: arsenate pKa1 ) 1.98; nitrate pKa1 ) -1.37; trifluoroacetate pKa1 ) 0.50; trichloroacetate pKa1 ) 0.52; dichloroacetate pKa1 ) 1.26; chloroacetate pKa1 ) 2.86; acetate pKa1 ) 4.76; chloride pKa1 ) -6.2; bromide pKa1 ) -9.0; iodide pKa1 ) -9.5. (Dean, J. A. Lange’s Handbook of Chemistry; McGraw-Hill Inc.; New York, 1992; pp 8.18-8.71).
substrate from the column. It was also assumed (as indeed was found subsequently by experiment) that increasing the pH of the buffering solution would cause an overall decrease in the times Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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Table 3. Affinity Constants for Phenyl-Substituted Acids and Neutral Sapphyrin in Methanola
Table 4. Elution Times for the Anion Forms of Various Benzoic and Benzenesulfonic Acid Derivatives
acid
Ka (M-1)b
substrate
pKa a
retention time (min)
phenylarsenic acid phenyl phosphonic acidc benzenesulfonic acid benzoic acid
1.9 × 105 1.7 × 104 320 102
anisic acidb benzoic acidb nitrobenzoic acidb
4.49 4.20 3.44
3.6 ((0.1) 3.8 ((0.1) 3.8 ((0.1)
sulfanilic acidc benzenesulfonic acidc 4-nitrophenylsulfonic acidc
3.25 2.55 1.74
3.2 ((0.1) 3.3 ((0.1) 3.3 ((0.1)
a
Affinity constants refer to the K11 values in ref 17a and correspond to the binding of free-base 3,12,13,22-tetraethyl-2,7,18,23-tetramethyl8,17-bis(hydroxypropyl)sapphyrin to the phenyl-substituted acid. The values given are the average of three repeat analyses; error estimate (15%. b The neutral dihydroxysapphyrin was added as 10 mM CD3OD solutions to 1 mM solutions of the phenyl-substituted acid in CD3OD with concentration changes being accounted for by EQNMR.21 In the determination of the stability constants, the possible effects of ion pairing (if any) were ignored. c Previously determined by 31P NMR spectroscopy.17a
needed to elute AMP from the sapphyrin-modified silica gel column. Such reductions in retention times would, it was presumed, simply reflect the experimentally reasonable proposition that the critical sapphyrin-AMP interactions would become weaker as the net concentration of monoprotonated species decreased. Inspection of Table 2 reveals that the AMP elution times are dependent upon the anion present in the mobile phase. Specifically, the data in this table reveal that elution times increase in the following order: arsenate > phosphate > chloride > sulfate > nitrate ) bromide > iodide > acetate. Such an elution order is consistent with the previous9a proposal that the extent of sapphyrin-oxyanion interaction parallels the relevant heteroatomto-oxygen bond distance. In the case of the halides, there is obviously no correlation between elution time and heteroatomto-oxygen bond length. In this case, the AMP elution times reflect, in reciprocal fashion, the known anion binding affinities, namely F- > Cl- = Br-, for the diprotonated form of 3,8,12,17,22hexaethyl-2,7,18,23-tetramethylsapphyrin in methanol.13 New Affinity Constant Determinations. Given the correlation between AMP elution times and sapphyrin halide affinities, it was reasonable to presume that a like correspondence would exist in the case of the oxyanion substrates. However, with the exception of those of certain phosphate anions, the relevant Ka values were not known at the outset of this work. Therefore, several affinity constants were newly measured as part of this work; the calculated Ka values, corresponding to the binding of anions to monoprotonated 3,12,13,22-tetraethyl-2,7,18,23-tetramethyl-8,17-bis(hydroxypropyl)sapphyrin in methanol, are given in Table 3. As can be deduced from an inspection of this table, the elution times recorded for AMP on the sapphyrin-modified silica gel do indeed correlate with these Ka values. The latter numbers, as expected, can also be correlated with heteroatomoxyanion bond length. Thus, there is a degree of internal agreement between Tables 2 and 3 that is both satisfying and reasonable. Tests of Generality. Sapphyrin-modified silica gels have previously been shown to be effective in allowing the HPLC-based separation of such classic phenyl-substituted anions as benzenesulfonate and benzoate.9a Left undetermined by this previous work was whether different anions of the same functional class (e.g., carboxylic acids) could be separated from one another. To address this issue, three benzoic acid and three sulfonic acid 2520 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
a Dean, J. A. Lange’s Handbook of Chemistry; McGraw-Hill Inc.: New York, 1992; pp 8.19-8.71 b Acetate anion elution, pH ) 7.0; for details, see text. c Sulfate anion elution, pH ) 7.0; for details see text.
Table 5. Times for AMP Elution from the Porphyrin-Bound Silica Gel Using Different Anionic Mobile Phases eluting anion (160 mM) phosphateb sulfateb nitrateb acetateb chlorideb
retention time (min) pH ) 6.0
pH ) 7.0
pH ) 8.0
3.8 ((0.1) 3.7 ((0.2) 3.2 ((0.08) 3.8 ((0.1) 3.9 ((0.05)
4.1 ((0.03) 3.9 ((0.09) 3.4 ((0.10) 4.5 ((0.08) 4.1 ((0.1)
4.6 ((0.07) 4.1 ((0.05) 3.7 ((0.06) 6.1 ((0.09) 4.2 ((0.04)
bond lengtha (Å) 1.56 1.49 1.28 1.26
a Pearson, W. B. Structure Reports; D. Reidel Publishing Co.: Boston, MA, 1981. b pKa’s of the conjugate acid forms of the anions contained in the mobile phase are as follows: phosphate pKa1 ) 2.1, pKa2 ) 7.20; sulfate pKa1 ) -3.1, pKa2 ) 1.98; nitrate pKa1 ) -1.37; acetate pKa1 ) 4.76; chloride pKa1 ) -6.2 (Dean, J. A. Lange’s Handbook of Chemistry; McGraw-Hill Inc.: New York, 1992; pp 8.18-8.71).
derivatives were separately tested using acetate and sulfate anion elution conditions, respectively. Unfortunately, as can be seen by inspection of Table 4, little in the way of useful within-category separation (e.g., sulfonate and benzoate) was achieved. Such findings, while disappointing, are not inconsistent with the basic hypothesis of this paper, namely that it is the heteroatom-tooxyanion distance, rather than anion size and shape, that determines the from-column elution order. Control Experiments with Porphyrin-Functionalized Silica Gel. As a further check that anion binding, as opposed to some other recognition phenomenon, is the critical determinant mediating anion retention selectivities in the case of the sapphyrin-based columns, an analogous porphyrin silica gel support was also prepared. Here, the synthesis was effected in accord with the method shown in Scheme 2 but using a porphyrin monocarboxylic acid23 instead of the corresponding sapphyrin precursor (see Experimental Section). In contrast to the sapphyrin-based system, the porphyrin functionalized “capped” silica gel was expected to provide a support that remains unprotonated at neutral pH.25 Not surprisingly, therefore, AMP elution experiments, analogous to those carried out with the sapphyrin silica gel supports, revealed little anion dependence (cf. Table 5). Further, raising the pH of the mobile phase actually increased the time required to elute AMP from this support. On this basis, we are inclined to believe that (25) Porphyrins are tetrapyrrolic, 18-π-electron, aromatic macrocycles that possess an inner core radius of 2.0 Å. This core size presumably prevents protonation at physiological pH as monoprotonated porphyrin is observed at pH phosphate > chloride > sulfate > nitrate ) bromide > iodide > acetate. This sequence is consistent with both previous chromatographic results9 and various solution phase Ka analyses,14,16,17,19 including some reported here for the first time. A wide range of control experiments, including those carried out with analogous porphyrin-based supports, serve to confirm the proposal that anion binding is indeed the critical predicate leading to both AMP binding and the above affinity sequence. Unfortunately, this study also served to reveal that the present sapphyrinderived silica gel is ineffectual in effecting the HPLC-based Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
2521
separations of mixtures of functionally analogous but structurally different anions (e.g, aromatic carboxylates). Current work in our laboratory is therefore focused on the study of silica gels bearing other pyrrole-derived anion-binding agents.10 ACKNOWLEDGMENT Financial support of this research by National Institutes of Health Grant AI33577, National Science Foundation Grant 9725399, and the Texas Advanced Technologies Program is gratefully acknowledged.
2522 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
SUPPORTING INFORMATION AVAILABLE Solution phase titration curves for the binding of phenylarsenic acid, benzenesulfonic acid, and benzoic acid to free-base 3,12,13,22-tetraethyl-2,7,18,23-tetramethyl-8,17-bis(hydroxypropyl)sapphyrin in methanol-d4 (3 pages). For ordering information, see any current masthead page. Received for review November 4, 1997. Accepted April 1, 1998. AC971214A
