Anal. Chem. 1998, 70, 2085-2091
Solid-Phase Lanthanide Luminescence Detection in Liquid Chromatography Thomas J. Wenzel,* Rogier Evertsen, Brooke E. Perrins, Thomas B. Light, Jr., and Amy C. Bean
Department of Chemistry, Bates College, Lewiston, Maine 04240
Terbium ions are immobilized on silica gel through ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) ligands. The resulting materials are utilized as solid-phase detection systems in liquid chromatography. Terbium(III) luminescence is observed when organic compounds that can transfer energy to Tb(III) by an intramolecular process are present. Peak tailing caused by slow dissociation of transferring compounds from the Tb(III) is significantly reduced if potassium acetate is added to the mobile phase. The solid phases slowly turn a bluish-green color unless triethylenetetraamine is added to the mobile phase. The color change is believed to be caused by bonding of trace amounts of transition metal ions to the solid phase. Detection of carboxylic acid-containing compounds such as indole-2-carboxylic acid, indole-3-acetic acid, 5-methoxyindole-2-carboxylic acid, kynurenic acid, and quinolinic acid is more sensitive with the Tb-DTPA phase than the Tb-EDTA phase, whereas detection of salicylic acid is more sensitive with Tb-EDTA than with Tb-DTPA. The use of time-resolved detection methods significantly enhances the sensitivity. Linearity, reproducibility, limits of detection, and chromatographic separations are examined for several compounds. Luminescent methods of detection are often used in liquid chromatography because of their sensitivity and selectivity. The lanthanide ions terbium(III) and europium(III) are unusual in their ability to undergo an energy transfer with certain excited-state organic compounds. The energy transfer occurs from the triplet state of the organic,1,2 and the efficiency depends on the match of the organic and lanthanide energy levels.3 Narrow-band emission from excited-state Tb(III) or Eu(III) is then observed. Energy transfer can occur by either an inter- or intramolecular process. Intramolecular transfer is preferable since the lanthanide is less susceptible to quenching by water or dissolved oxygen.4,5 The method is especially selective as only certain functionalities, notably benzoyl groups or nitrogen heterocycles, have triplet-state (1) Crosby, G. A.; Whan, R. E.; Alire, R. M. J. Chem. Phys. 1961, 34, 744. (2) Crosby, G. A.; Whan, R. E.; Freeman, J. J. J. Phys. Chem. 1962, 66, 2493. (3) Sinha, A. P. B. In Spectroscopy in Inorganic Chemistry; Rao, C. N. R., Ferraro, J. R., Eds.; Academic Press: New York, 1971; Vol. 2, p 255. (4) DiBella, E. E.; Weisman, J. B.; Joseph, M. J.; Schultz, J. R.; Wenzel, T. J. J. Chromatogr. 1985, 328, 101. (5) Wenzel, T. J.; Collette, L. M.; Dahlen, S. T.; Hendrickson, S. M.; Yarmaloff, L. W. J. Chromatogr. 1988, 433, 149. S0003-2700(97)01269-9 CCC: $15.00 Published on Web 04/10/1998
© 1998 American Chemical Society
energies that match those of the excited states of Eu(III) or Tb(III). The use of lanthanide ions as luminescent detection chromophores in liquid chromatography was first described by DiBella et al.4 for the detection of aromatic ketones and aldehydes. The method has since been extended to a number of other compounds including tetracyclines,5,6 single-stranded nucleic acids,7 nalidixic acid and bleomycins,8 indoles and heterocyclics,9 orotic acid,10,11 steroids such as progesterone and testosterone,12,13 diphacinone,14 fluoroquinolines,15 propyl gallate,16 ochratoxin A and citrinin,17 theopylline,18 amino compounds,19 labeled thiols,20,21 and hydrolysis products of dexrazoxane.22 Inorganic ions have been measured using quenched lanthanide luminescence techniques.23,24 The applicability of lanthanide luminescence detection in electrophoretic techniques has also been described.11,13,25 All prior applications of lanthanide luminescence detection in liquid chromatography have involved either pre- or postcolumn addition of the lanthanide ion to the mobile phase. Postcolumn addition is advantageous since the lanthanide ion cannot influence the separation. Also, the postcolumn conditions can be adjusted to optimize detection sensitivity. Such an adjustment was beneficial in the detection of tetracycline with Eu(III), in which (6) Duggan, J. X. J. Liq. Chromatogr. 1991, 14, 2499. (7) Wenzel, T. J.; Collette, L. M. J. Chromatogr. 1988, 436, 299. (8) Wenzel, T. J.; Zomlefer, K.; Rapkin, S. B.; Keith, R. H. J. Liq. Chromatogr. 1995, 18, 1473. (9) Schreurs, M. Ph.D. Thesis, Free University, Amsterdam, 1992. (10) Schreurs, M.; Vissers, J. P. C.; Gooijer, C.; Velthorst, N. H. Anal. Chem. Acta 1992, 262, 201. (11) Milofsky, R. E.; Spaeth, S. Chromatographia 1996, 42, 12. (12) Amin, M.; Harrington, K.; von Wandruszka, R. Anal. Chem. 1993, 65, 2346. (13) Milofsky, R. E.; Mahlberg, M. G.; Smith, J. M. J. High. Resolut. Chromatogr. 1994, 17, 731. (14) Panadero, S.; Gomez-Hens, A.; Perez-Bendito, D. Anal. Chim. Acta 1993, 280, 163. (15) Rieutord, A.; Vazquez, L.; Soursac, M.; Prognon, P.; Blais, J.; Bourget, P.; Mahuzier, G. Anal. Chim. Acta 1994, 290, 215. (16) Panadero, S.; Gomez-Hens, A.; Perez-Bendito, D. Analyst 1995, 120, 125. (17) Vazquez, B. I.; Fente, C.; Franco, C.; Cepeda, A.; Prognon, P.; Mahuzier, G. J. Chromatogr. 1996, 727, 185. (18) Mwalupindi, A. G.; Warner, I. M. Anal. Chem. Acta 1995, 306, 49. (19) Iwata, T.; Senda, M.; Kurosu, Y.; Tsuji, A.; Maeda, M. Anal. Chem. 1997, 69, 1861. (20) Schreurs, M.; Gooijer, C.; Velthorst, N. H. Anal. Chem. 1990, 62, 2051. (21) Schreurs, M.; Hellendoorn, L.; Gooijer, C.; Velthorst, N. H. J. Chromatogr. 1991, 552, 625. (22) Hasinoff, B. B. J. Chromatogr., B. Biomed. Appl. 1994, 656, 451. (23) Schreurs, M.; Somsen, G. W.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1989, 482, 351. (24) Baumann, R. A.; Kamminga, D. A.; Derlagen, H.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1988, 439, 165. (25) Rieutord, A.; Prognon, P.; Mahuzier, G. Analusis 1996, 24, 349.
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chromatographic separation was best achieved at acidic pH whereas detection sensitivity was optimized at basic pH.5 Postcolumn addition dilutes the column effluent, however, which raises detection limits. Also, noise from pulsing of the postcolumn pump is often the limiting factor in achievable limits of detection. There are restrictions on the conditions under which lanthanide ions can be employed in either pre- or postcolumn mode. Hydrated lanthanide(III) ions, as observed with solutions of nitrate or chloride salts, form insoluble precipitates at pH values of 7 or higher. Lanthanide ions also form insoluble salts with phosphate species. Addition of an encapsulating ligand such as ethylenediaminetetraacetic acid (EDTA) can be used to solubilize lanthanide ions at basic pH or in the presence of phosphate buffers.5 Since the EDTA ligand does not fully surround the lanthanide metal, direct binding of transferring molecules to the lanthanide can still occur. An alternative to pre- or postcolumn addition is to utilize a solidphase lanthanide detection system. In such a system, the lanthanide must be immobilized on a suitable solid support, and the lanthanide-containing solid is then placed in the detector flow cell. In this report, we describe the immobilization of lanthanide ions on silica gel through the use of covalently bound EDTA or diethylenetriaminepentaacetic acid (DTPA) moieties. Preliminary investigations of the conditions under which these phases can be used for detection in liquid chromatography are presented. To our knowledge, this is the first application of solid-phase lanthanide luminescence detection in liquid chromatography. EXPERIMENTAL SECTION Reagents. Silica gel (Davisil 60/100 mesh), (aminopropyl)triethoxysilane, ethylenediaminetetraacetic dianhydride, diethylenetriaminepentaacetic dianhydride, anhydrous methyl sulfoxide, ethylenediamine (EN), triethylenetetraamine (TRIEN), tetrabutylammonium bromide, potassium acetate, and [bis-2-hydroxyethyl)amino]tris(hydroxymethyl)methane (BIS-TRIS), were obtained from Sigma-Aldrich, Milwaukee, WI. Silica gel (5-µm dp with aminopropyl surface groups) was obtained from Alltech Associates, Deerfield, IL. Terbium(III) chloride or nitrate was prepared using Ultrex hydrochloric or nitric acid (J. T. Baker, Phillipsburg, PA) as described.4 Indole-2-carboxylic acid, indole3-acetic acid, 5-methoxyindole-2-carboxylic acid, kynurenic acid, salicylic acid, and quinolinic acid, which are known to undergo an intramolecular energy transfer with lanthanides, were obtained from Sigma-Aldrich). Apparatus. Luminescence measurements were performed using a Perkin-Elmer LS-5 fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT) equipped with a pulsed xenon lamp (60 Hz with pulse width less than 10 µs) and a model 3700 Data Station. Excitation and emission slits were set to 10 nm (wavelength accuracy to 2 nm, nominal band-pass of 10 nm) when measurements were performed in the flow mode. The response time was 0.5 s. Tb(III) luminescence was detected at an emission wavelength of 545 nm. The following excitation wavelengths were used: salicylic acid, 327 nm; kynurenic acid, 300 or 339 nm; quinolinic acid, 300 or 339 nm; indole-3-acetic acid, 285 or 300 nm; indole-2-carboxylic acid, 300 nm; and 5-methoxyindole-2carboxylic acid, 285 or 300 nm. Wavelength programming was employed to optimize the sensitivity during liquid chromatographic separations. For time-resolved measurements, the instru2086 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
Scheme 1
ment was set in the phosphorescence mode and appropriate delay (td) and gate (tg) times were entered. Flow injection and liquid chromatographic analyses were performed using a Beckman 110 B solvent delivery module (Beckman Instruments, Berkeley, CA), Lo-pulse model LP-21 Pulse Dampener (Scientific Systems, State College, PA), an injection valve equipped with a 20-µL loop, and an Econosil C-18 column (10 µm, 250 mm × 4.6 mm; Alltech, Deerfield, IL). The flow rate was 0.5 mL/min for flow injection studies and 1.0 mL/ min for liquid chromatographic separations. Diffuse reflectance infrared spectra were obtained on a PerkinElmer model 16 PC Fourier transform infrared spectrophotometer. Synthesis. Immobilization of lanthanide ions on silica gel was achieved using the procedure in Scheme 1. Preparation of Aminopropyl-Derivatized Silica Gel (SGNH2). Underivatized silica gel was activated by refluxing in concentrated hydrochloric acid for 4 h.26 The silica gel was collected in a fritted glass filter and washed with water until the washings were neutral. It was then dried at 220 °C for 4 h. Activated silica gel (14.4 g) and a solution of (3-aminopropyl)triethoxysilane (12.2 g, 55 mmol) in 115 mL of dry toluene were added to a 250-mL, three-necked, round-bottomed flask fitted with a condenser and a nitrogen purge. The mixture was gently stirred and heated to 60 °C for 4 h. After cooling to room temperature, the derivatized silica gel was isolated by suction filtration on a glass filter and washed with 100 mL each of toluene, chloroform, and methanol. The product was then dried overnight in an oven at 80 °C. Coupling of DTPA to Aminopropyl Silica Gel (SG-DTPA). Coupling of DTPA to the aminopropyl silica gel was accomplished by a modification of a procedure used to couple DTPA to other amines.27 SG-NH2 (2.0 g, 2.4 mmol of amino group based on elemental analysis data) and a solution of DTPA dianhydride (1.2 g, 3.3 mmol) in 20 mL of dry dimethyl sulfoxide (DMSO) were added to a 50-mL, three-necked, round-bottomed flask fitted with a condenser and a nitrogen purge. The mixture was gently stirred for 4 h, after which the silica gel was isolated by suction filtration on a glass filter and washed with 60 mL of DMSO. The silica gel was transferred to a beaker and gently stirred in 60 mL of distilled water for 30 min. After collection by suction filtration on a glass filter, and washing with 60 mL of methanol, the product was dried (26) Aue, W. A.; Hastings, C. J. J. Chromatogr. 1969, 42, 322 (27) Bailey, M. P.; Rocks, B. F.; Riley, C. Analyst 1984, 109, 1449.
at 80 °C for 24 h. Preparation of the desired material was confirmed by infrared spectroscopy and elemental analysis. Coupling of EDTA to Aminopropyl Silica Gel (SG-EDTA). SG-EDTA was prepared by a procedure identical to that used to prepare SG-DTPA except that EDTA dianhydride (1.0 g, 3.9 mmol) was reacted with 2.0 g of SG-NH2. Preparation of the desired material was confirmed by infrared spectroscopy and elemental analysis. Preparation of the Tb(III) Complex of SG-DTPA (SGDTPA-Tb) and SG-EDTA (SG-EDTA-Tb). SG-DTPA (0.50 g) and 130 mL of 0.010 M sodium bicarbonate were combined in a beaker, gently stirred, and warmed to 50 °C for 30 min. The sodium form of SG-DTPA (SG-DTPA-Na) was collected by suction filtration on a glass filter and washed with 200 mL of distilled water and 100 mL of methanol. The SG-DTPA-Na was transferred to a beaker containing a solution of Tb(NO3)3‚6H2O (0.24 g, 0.50 mmol) in methanol (25 mL) and gently stirred for 30 min. The product (SG-DTPA-Tb) was collected by suction filtration on a glass filter, washed with 100 mL of methanol, and dried overnight at 80 °C prior to use. SG-EDTA-Tb was prepared by an identical procedure except that SG-EDTA was used in place of SG-DTPA. Flow Cell Design. A small-volume flow cell for use with solidphase detection systems has been described in the literature.28 For the preliminary analysis conducted herein, a simpler design was utilized. Glass (2 mm i.d.) or quartz (either 2 or 1 mm i.d. and 3 mm o.d.) tubing (Quartz Scientific, Fairport Harbor, OH) was cut into lengths of ∼90 mm. The cell was packed with approximately 15-20 mm of Tb-loaded silica gel that was held in place at both ends with plugs of glass wool. The packing was placed in the tubing to minimize dead volume at the entry side. Stainless steel tubing (1/16 in.) from the liquid chromatographic column was attached to the glass cell with a 1/16-1/8-in. stainless steel Swagelok union. Supeltex M-1 ferrules (Supelco, Bellefonte, PA) were used on the end with the glass cell. The flow cell was mounted in the proper location relative to the excitation beam and emission slit of the fluorescence spectrofluorometer by modifying a cuvette holder. The glass wool plugs retained the larger particle (60/100 mesh) phase in the cell. With the smaller particles (5 µm), the packing gradually moved down the tube. The use of smaller particle phases for an extended time period would require a design with a rigid, porous frit to hold the particles in place. Since the sensitivity was comparable for the large- and small-particle phases, the majority of measurements were performed on the larger particle phase. The best limits of detection were obtained with 1-mm-i.d. quartz tubes. Mobile Phases. All mobile phases were prepared using water that was first distilled and then purified by passage through a Milli-Q deionization device and HPLC-grade methanol or acetonitrile. Mobile phases were mixed on a volume-volume basis, filtered through an 0.45-µm membrane filter, and sonicated for 15 min prior to use. Stock solutions of energy-transferring compounds were prepared by weight and diluted to the necessary concentration using the appropriate mobile phase. Solutions of (28) Van Zoonen, P.; Kamminga, D. A.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1986, 58, 1245.
Figure 1. Diffuse reflectance infrared spectra of (a) activated silica gel, (b) SG-EDTA, and (c) SG-EDTA-Tb.
the transferring compounds were sonicated for 15 min prior to use. Procedures. Preliminary investigations of the solid phase detection system were conducted using flow-injection analysis (FIA). The system was equilibrated by pumping the mobile phase through the solid phase for 30 min. In some instances, a solution of the organic compound dissolved in the same mobile phase was then pumped for 10 min, followed by a 30 min flow of mobile phase. The cycle was repeated two more times, after which the mobile phase was pumped for an additional 90 min to complete data collection and reequilibrate the system. In other instances, only 20 µL increments of solutions of the transferring compound were injected using the valve and sample loop. RESULTS AND DISCUSSION: Preparation of Solid Phases. Coupling of the EDTA or DTPA unit to SG-NH2 is confirmed by infrared spectroscopy and elemental analysis. The infrared spectra of SG-EDTA and SGDTPA (Figure 1b) show a new band at 1725 cm-1 that is characteristic of the carbonyl stretch of a carboxylic acid.29 The carbonyl stretch of carboxylate ions is typically observed between 1650 and 1550 cm-1,29 and the band at 1725 cm-1 is no longer observed when the sodium salt or lanthanide(III) complexes of the phases are prepared (Figure 1c). Elemental analyses of SGNH2 we prepared (C, 5.2%, N, 1.7%) indicate a coverage of ∼1.2 mmol/g of aminopropyl functionality (0.017 g of nitrogen or 0.001 19 mol/g of silica gel). Assuming the only carbon and nitrogen in the derivatized silica gel comes from the aminopropyl functionality, for a nitrogen value of 1.7%, the expected carbon value is 4.37%. The additional carbon may come from ethoxy groups that have not been hydrolyzed. Elemental analyses of commercially available aminopropyl-derivatized silica gel also showed higher than expected carbon values. Elemental analyses (29) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 5th ed.; Wiley: New York, 1997; pp 117-118.
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Figure 2. FIA of indole-2-carboxylic acid (10-4 M) in a mobile phase consisting of (a) 0.01 M BIS-TRIS, 10-4 M EDTA (pH 6)-methanol (50:50 v/v) and (b) 0.01 M BIS-TRIS, 10-4 M EDTA, 0.20 M potassium acetate (pH 6)-methanol (50:50 v/v). Detection phase, SG-EDTATb.
of SG-DTPA (C, 10.1%, N, 3.1%) and SG-EDTA (C, 10.0%, N, 2.7%) are both consistent with derivatization of just under half of the amino groups and a surface coverage of 0.5 mmol/g based on nitrogen and carbon analysis. Assuming hydrolysis of all remaining ethoxy groups during the coupling of DTPA to the aminopropyl silica gel, calculated nitrogen and carbon analyses for 0.7 mmol/g aminopropyl (C3H8N) and 0.5 mmol/g aminopropyl-DTPA (additional C14H22N3O9 for each derivatized functionality) are 3.2 and 10.8%, respectively. Calculated nitrogen and carbon values for 0.7 mmol of aminopropyl and 0.5 mmol of aminopropyl-EDTA (additional C10H15N2O7 for each derivatized functionality) are 2.7 and 9.1%, respectively. Surface coverages of ∼0.5 mmol/g of DTPA or EDTA are consistently obtained from replicate syntheses. Mixtures of the terbium-loaded phases with solutions of indole-2-carboxylic acid, indole-3-acetic acid, 5-methoxyindole-2-carboxylic acid, kynurenic acid, salicylic acid, or quinolinic acid exhibit an intense lanthanide luminescence when irradiated with long-wavelength ultraviolet light. The same is observed when solutions of nalidixic acid are added to a europiumcontaining phase. Solid-phase detection should therefore be generally applicable to the range of compounds that have been previously detected using solution-phase lanthanide luminescence detection. Mobile-Phase Additives. Severely tailed peaks, as seen in Figure 2a, are observed in mobile phases consisting only of methanol-water. The tailing is independent of the concentration of injected material, indicating that it is caused by slow dissociation of the transferring compound from the solid-phase Tb(III), rather than overloading of the phase. The tailing is significantly reduced by addition of potassium acetate to the mobile phase, as seen in Figure 2b. Acetate ion exhibits a relatively weak inner-sphere complexation with lanthanide ions,30-33 and serves to compete with and displace the energy-transferring compound from the lanthanide. Tailing is reduced at concentrations up to 0.20 M (30) Spiro, T. G.; Revesz, A.; Lee, J. J. Am. Chem. Soc. 1968, 90, 4000. (31) Reuben, J.; Fiat, D. J. Chem. Phys. 1969, 51, 4909. (32) Choppin, G. R.; Henrie, D. E.; Buijs, K. Inorg. Chem. 1966, 5, 1743. (33) Kropp, J. L.; Windsor, M. W. J. Phys. Chem. 1967, 71, 477.
2088 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
Figure 3. FIA of salicylic acid (10-4 M) in a mobile phase consisting of (a) 0.20 M potassium acetate-methanol (50:50 v/v) and (b) 0.2 M potassium acetate, 10-5 M TRIEN (pH 7.9)-methanol (50:50 v/v). Detection phase, SG-EDTA-Tb.
potassium acetate with no loss of, or even a slight improvement in, sensitivity. In previous work using lanthanide luminescence detection, the addition of potassium acetate caused significant improvements in sensitivity with compounds that transfer energy by an intermolecular process.4 It was proposed that the acetate ion displaces water from the first coordination sphere, thereby reducing quenching, but does not bind so strongly as to inhibit energy-transferring collisions. The association of acetate ion with lanthanide ions is not sufficiently large to eliminate binding of the energy-transferring compounds examined in our study. The retention of, or slight improvement in, sensitivity suggests that the acetate ion also displaces water from the first coordination sphere of the lanthanide ion. Mobile phases containing potassium acetate, sodium phosphate, or salicylic acid slowly turn the head of the solid phase a bluish-green color. Methanol-water mixtures alone do not cause the color change. Furthermore, the signal from an energytransferring compound diminishes in intensity upon replicate injections when using these phases (Figures 2 and 3a). The discoloration is believed to be caused by transition metal ions such as Cu(II) and Ni(II) that are present as impurities in the mobilephase constituents and/or leached from metal surfaces in the chromatographic system. Trace amounts of transition metal ions would associate with the bonded EDTA or DTPA moieties and gradually displace the Tb(III), thereby diminishing the intensity of energy-transferring compounds upon replicate injections. Amine-containing compounds such as EN and TRIEN are chelating ligands that have high association constants with many transition metal ions but low association constants with lanthanides.34 At the pH employed (6.5-7.5), EN (pK1, 6.85; pK2, 9.93 for H2EN2+) exists primarily as a mixture of its mono- and diprotic forms, neither of which is a suitable chelating ligand. Addition of EN to the mobile phase does not eliminate the discoloration. TRIEN (pK1, 3.32; pK2, 6.67; pK3, 9.20; pK4, 9.92 for H4TRIEN4+) exists as a mixture of its di- and triprotic forms (34) Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum Press: New York, 1974-1982; Vols. 1-5.
at pH 6.5-7.5, and the diprotic form is still a chelating ligand. Addition of TRIEN to the mobile phase eliminates the discoloration. Furthermore, the intensity of Tb(III) luminescence does not diminish with replicate injections of salicylic acid (Figure 3b) or as a solution of salicylic acid is continually pumped through the solid phase. The solid-phase system can be used under such conditions for many hours without any diminishment of signal. Reproducibility is excellent as relative standard deviations of ∼1.5% are typically observed for five replicate injections of transferring compounds. SG-EDTA-Tb vs SG-DTPA-Tb. It is essential that the lanthanide not be displaced by components of the mobile phase in a solid-phase detection system. Since lanthanide complexes are primarily ionic in nature and kinetically labile, it is necessary to use a chelating ligand with a large formation constant. EDTA and DTPA were selected as immobilizing ligands since they form particularly stable complexes with lanthanide ions.35 The immobilizing ligand must not fully encapsulate the lanthanide ion, otherwise intramolecular energy transfer will not be possible. The extent to which the lanthanide ion is encapsulated, though, is significant. Water effectively quenches lanthanide luminescence when bound in the first coordination sphere.33,36,37 If the immobilizing ligand does not occupy enough of the coordination sites, water molecules and transferring compounds may simultaneously bind, thereby reducing the intensity of luminescence. Crystal structures of lanthanide-EDTA complexes indicate that the ligand only surrounds about half of the metal ion and that three or four waters of hydration are in the first coordination sphere.38 The larger DTPA ligand is more effective at encapsulating lanthanide ions, which accounts for the utility of Gd(DTPA)2complexes as NMR relaxation reagents.39 The DTPA ligand does not fully surround the metal ion, though, and one water molecule associates in the first coordination sphere of the lanthanide both in solid complexes40 and in solution.41 Conversion of one of the carboxylate units of EDTA or DTPA into an amide group, as occurs with the solid phases described herein, does lower the formation constant with lanthanide metals (1022 vs 1019 for DTPA35 and DTPA-amide42 complexes). However, conversion of two carboxylate groups into amide groups does not reduce the extent to which the DTPA ligand encapsulates the metal and does not eliminate the presence of one water molecule in the first coordination sphere of the lanthanide ion.43 For salicylic acid, which associates with lanthanides in a bidentate manner that forms a six-membered chelate ring, the signal-to-noise ratio is ∼5 times larger with the SG-EDTA-Tb (25:1) than with SG-DTPA-Tb (5:1) (Figure 4). For compounds with a single carboxylate group such as indole-2-carboxylic acid and 5-methoxyindole-2-carboxylic acid, which presumably associ(35) Moeller, T.; Martin, D. F.; Thompson, L. C.; Ferrus, R.; Feistel, G. R.; Randall, W. J. Chem. Rev. 1965, 65, 1. (36) McCarthy, W. J.; Winefordner, J. D. Anal. Chem. 1966, 38, 848. (37) Matovich, E.; Suzuki, C. K. J. Chem. Phys. 1963, 39, 1442. (38) Lind, M. D.; Lee, B.; Hoard, J. L. J. Am. Chem. Soc. 1965, 87, 1611. (39) Wenzel, T. J.; Ashley, M. E.; Sievers, R. E. Anal. Chem. 1982, 54, 615. (40) Gries, H.; Miklautz, H. Physiol. Chem. Phys. Med. NMR 1984, 16, 105. (41) Jenkins, B. G.; Lauffer, R. B. Inorg. Chem. 1988, 27, 4730. (42) Lauffer, R. B.; Brady, T. J. Magn. Reson. Imaging 1985, 3, 11. (43) Konings, M. S.; Dow, W. C.; Love, D. B.; Raymond, K. N.; Quay, S. C.; Rockledge, S. M. Inorg. Chem. 1990, 29, 1488.
Figure 4. FIA of salicylic acid (10-4 M) in a mobile phase consisting of 0.20 M potassium acetate-methanol (50:50 v/v) using (a) SGDTPA-Tb and (b) SG-EDTA-Tb.
ate in a bidentate manner that forms a four-membered chelate ring, an ∼3-fold improvement in sensitivity is observed with SGDTPA-Tb than with SG-EDTA-Tb. These observations suggest that the DTPA-amide ligand does not encapsulate Tb(III) enough to prevent association and intramolecular energy transfer with carboxylate compounds, but does prevent association with compounds such as salicylic acid that form larger chelate rings. The reduced sensitivity for carboxylate compounds with the SGEDTA-Tb compared to SG-DTPA-Tb suggests that water molecules simultaneously bind to Tb in the EDTA phase and partially quench the signal. The trends are consistent enough so that the SG-EDTA-Tb is recommended for use with chelating compounds that form six-membered or larger rings, whereas SGDTPA-Tb is recommended for compounds that only bind through a single carboxylate moiety. Time-Resolved Measurements. Lanthanide ions such as Tb(III) and Eu(III) have long excited-state lifetimes because the luminescent transitions are spin forbidden. Time-resolved measurements can enhance the sensitivity of lanthanide luminescence detection because the reduction in noise caused by elimination of background fluorescence and scatter more than offsets the drop in lanthanide luminescence.7,8 Background fluorescence and scatter are significant in the solid-phase lanthanide systems as seen by measuring the luminescent response at wavelengths near 545 nm as a function of delay time (Figure 5). A delay time of 0.04 ms eliminates most of the scatter and background fluorescence. Using a delay time of 0.04 ms, gate times between 0.75 and 1.5 ms provide the best signal-to-noise ratio. Figure 6 shows the response for replicate injections of salicylic acid with continuous and time-resolved measurements. The approximately 6-fold improvement in the signal-to-noise ratio (9.7:1 in the continuous mode, 62:1 in the time-resolved mode) is typical for the compounds tested. Compatibility with Mobile Phases and Chromatographic Results. The solid-phase detection system is compatible with a variety of mobile phases, including those containing water, methanol, and acetonitrile. Weak lanthanide-complexing ligands such as nitrogen bases and acetate ion do not strip Tb(III) from the solid phase. BIS-TRIS can be used as a buffer, and tetrabutylammonium bromide can be used as an ion interaction reagent. A mixture of indole-3-acetic acid, indole-2-carboxylic acid, and 5-methoxyindole-2-carboxylic acid was separated on a Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
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Figure 5. (a) Emission spectra at various delay times: top, 0.01 ms; middle, 0.02 ms, bottom, 0.05 ms. (b) Luminescence response at different emission wavelengths and delay times. Solution, salicylic acid (2.3 × 10-4 M) in 0.2 M potassium acetate, 10-5 M TRIEN (pH 7.9)-methanol (50:50 v/v); detection phase, SG-EDTA-Tb; gate time, 1.00 ms.
Figure 6. FIA of salicylic acid (7.1 × 10-6 M) in a mobile phase consisting of 0.2 M potassium acetate, 10-5 M TRIEN (pH 7.9)methanol (50:50 v/v) recorded in (a) fluorescence and (b) phosphorescence mode: td, 0.04 ms; tg, 1.00 ms. Detection phase, SGEDTA-Tb.
C-18 column using a mobile phase of 0.01 M sodium phosphate, 10-5 M TRIEN, 10-3 M tetrabutylammonium bromide (pH 7.5)methanol (72.5:27.5 v/v). Minimal peak tailing was noticed, indicating that phosphate acts similarly to acetate and displaces the analytes from Tb(III). Unfortunately, the sensitivity was also reduced when compared to mobile phases with potassium acetate and BIS-TRIS buffers, indicating that phosphate may associate too strongly and inhibit binding of the transferring molecule. 2090 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
Figure 7. Separation of indole-3-acetic acid (1), 5-methoxyindole2-carboxylic acid (2), and indole-2-carboxylic acid (3) in a mobile phase consisting of 0.01 M BIS-TRIS, 10-3 M tetrabutylammonium bromide, 0.20 M potassium acetate, 10-5 M TRIEN (pH 7.5)methanol (72.5:27.5 v/v). Detection phase, SG-DTPA-Tb in quartz tubing (1.0 mm i.d.); td, 0.04 ms; tg, 1.00 ms; emission wavelength, 545 nm; slit widths, 10 nm; and excitation wavelength of (a) 300 nm and (b) 285 nm.
The solid-phase system can be used for chromatographic detection as seen by the chromatograms of indole-3-acetic acid, indole-2-carboxylic acid, and 5-methoxyindole-2-carboxylic acid shown in Figure 7. The retention times we observe are comparable to those previously reported,9 indicating that the relatively high concentration of potassium acetate does not influence the retention of these compounds. The chromatograms in Figure 7 also illustrate the importance of optimizing the excitation wavelength. Limits of detection for these three compounds using solidphase detection are 2 × 10-5 M for indole-3-acetic acid, 2.2 × 10-6 M for 5-methoxyindole-2-carboxylic acid, and 1.7 × 10-6 M for indole-2-carboxylic acid. Linear response is observed over at least 2 orders of magnitude for all of the compounds employed in this study. The limits of detection with the solid-phase system are ∼1 order of magnitude higher in concentration that those previously reported using postcolumn addition of Tb(III);9 however, the mass limit of detection for the compounds was only ∼3 times higher than the previous report in which a larger volume injection loop was used. Furthermore, the solid-phase flow cell employed in this study is not nearly an optimized system. Optimization of a solid-phase detection system by minimizing both the dead volume and flow cell volume is expected to improve the detection limits considerably. CONCLUSION Lanthanide ions such as Tb(III) or Eu(III) can be immobilized on silica gel through bonded EDTA or DTPA moieties and used for solid-phase luminescence detection in liquid chromatography. Severe tailing of energy-transferring compounds is observed due to slow dissociation of the compounds from the lanthanide ion. The tailing can be reduced by adding potassium acetate to the
mobile phase. Trace amounts of transition metal ions in the mobile phases are believed to cause a slow discoloration of the solid phase and reduction in signal intensity over time. Addition of TRIEN to mobile phases eliminates the discoloration and leads to reproducible response. Detection of carboxylate compounds is more sensitive with the Tb-DTPA phase whereas detection of a chelating compound such as salicylic acid is more sensitive with the Tb-EDTA phase. Finally, sensitivity is enhanced using timeresolved methods.
ACKNOWLEDGMENT We appreciate the support for T.B.L. through The Camille and Henry Dreyfus Foundation (Special Grant in support of secondary school science teachers). This work was funded in part by a grant to Bates College from the Howard Hughes Medical Institute. Received for review November 19, 1997. Accepted March 4, 1998. AC971269C
Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
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