2022
Anal. Chem. 1982, 5 4 , 2022-2025
(4) Lloyd, J. B. F. J . Forensic Sci. Soc. 1971, 2 , 235-253. (5) Vo-Dinh, T.; Mortinez, P. R. Anal. Chlm. Acta 1981, 125, 13-19. (6) Eastwood, D.; Fortler, S. H.; Hendrick, M. S. Am. Lab. (Fairfield, Conn.) 1978, 10, 45-51. (7) John, P.; Soutar, I., Anal. Chem. 1976, 48, 520-524. (8) Lloyd, J. B. F. Analyst (London) 1980, 105,97-109. (9) Wakeham, S. 0. Envlron. Scl. Technol. 1977, 7 1 , 272-276. (10) Vo-Dinh, T.; Gammage, R. B.; Martinez, P. R. Anal. Chem. 1981, 53, 253-258. (11) Vo-Dinh, T.; Gammage, R. B.; Hawthorne, A. R.; Thorngale, J. H., Envlron. Sci. Technol. 1978, 12, 1297-1302. (12) Andre, J. C.; Baudot, Ph.; Nlciause, M. Clin. Chlm. Acta 1977, 7 6 , 55-66. (13) Weiner, E. R. Anal. Chem. 1978, 50, 1583-1584. (14) Johnson, D. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1977, 49, 747A-757A.
(15) Warner,-I. M.; Christian, G. D.; Davidson, E. R.; Callis, J. B. Anal. Chem. 1977, 49, 564-573. (16) Ho, C.-N.; Christian, G. D.; Davidson, E. R. Anal. Chem. 1981, 53, 92-98. (17) Ho, C.-N.; Christian, G. D.; Davidson, E. R. Ana/. them. 1978, 50, 1 108-1 1 13.
(18) Ho, C.-N.; Christian, G. D.; Davidson, E. R. Anal. Chem. 1980, 52, 107 1-1 079. (19) Jursensen, A.; Inman, E. L.. Jr.; Winefordner, J. D. Anal. Chim. Acta 1961, 137, 187-194. (20) Rho, J. H.; Stuart, J. L. Anal. Chem. 1978, 50,620-625. (21) HO, C.-N.; Warner, I. M.; Fogarty, M. P. Ind. Res. Dev. 1981, 23, 118-122 . .- . (22) Inman, E. L., Jr.; Winefordner, J. D., unpublished work. (23) Vo-Dinh, T. Anal. Chem. 1978, 5 0 , 396-401. (24) Andre, J. C.; Bouchy, M.; Virlot, M. L. Anal. Chim. Acta 1979, 105, 297-310. (25) Lloyd, J. B. F.; Evett, I.W. Anal. Chem. 1977, 4 9 , 1710-1715. (26) Hershberger, L. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1981, 53, 971-975. (27) Vo-Dlnh, T.; Gammage, R. B. Anal. Chem. 1978, 50, 2054-2058.
RECEIVED for review December 28, 1981. Accepted July 6, 1982. This work Was supported by "-I-GM11373-19
DOE-DE-ASOJ-78EV06022AOO2.
and
Spectrofluorometric Determination of Calcium and Lanthanide Elements in Dilute Solution Theodore L. Miller" and Stuart I. Senkfor Department of Chemistry, Ohio Wesleyan University, Delaware, Ohio 430 15
Nonradlative energy transfer from the ligand diplcoilnic acid (DPA) to ianthanlde Ions has been used to develop a qulck, easy, and reliable method to determine calcium and lanthanide elements in dllute aqueous solution. Tb( I I I), Eu( I I I), and Dy(II1) are determined dlrectly and large useful concentratlon ranges are avallabie for quantitative analysls. When other metal ions are added to a solutlon of Tb(DPA)?-, the terbium lumlnescence Is reduced. At low metal Ion concentrations, the reductlon is linear for calclum and lanthanide Ions and can be used as an anaiytlcai callbration curve. The detection llmlt Is about 0.1 pg/L for the ions. The method Is readlly adaptable to microsampilng and caiclum can be determlned In the presence of magnesium without Interference.
Lanthanide ions have become an important tool as luminescent probes in biological systems (1-3) and for excited molecules in aqueous solution (4).As research continues and applications increase, a quick, easy, and reliable analytical method for measuring trace lanthanide ion concentrations becomes a necessity. Absorptions measurements are possible but only give satisfactory resulb for high lanthanide ion concentrations because the molar absorptivities of the ions are rather low (5). Spectrophotometric reagents (6-8) have been used to increase the sensitivity. Atomic absorption spectrometry (9) yields sensitivities not much greater than the methods based on spectrophotometric reagents since the absorptivity of the lanthanide elements is low. A promising alternative in both sensitivity and selectivity is luminescence spectrometry. In aqueous solutions high concentrations of Gd(III), Tb(III), and Eu(II1) emit with moderate strength; the emission from Sm(111)and Dy(II1) is weaker, and the other ions display little or no luminescence (10). Therefore, to develop a spectrofluorometric method for the determination of trace amounts of trivalent lanthanide ion, either the use of a high-intensity laser excitation source (analytical determinations using a
conventional spectrophotofluorometer are not possible) or excitation of the lanthanide ions by a nonradiative energy transfer process is required. Intermolecular (11) and intramolecular (12) energy transfer have both been investigated. This paper describes the use of intramolecular energy transfer from pyridine-2,6-dicarboxylicacid (dipicolinic acid-DPA) in the spectrofluorometric determination of calcium and lanthanide elements in dilute solution.
EXPERIMENTAL SECTION Apparatus. Absorption spectra were recorded at room temperature on a Cary 219 spectrophotometer by Varian using matched quartz cells with a 1-cm pathlength. All the luminescent measurements were made with an Aminco-Bowman Ratio I1 spectrofluorometer at room temperature. Either a standard 1 cm square quartz cell or a new microcell designed in our laboratory (the specific design and performance of the microcell will be discussed in a separate publication) was used with the spectrofluorometer. Reagents. The lanthanide salts were all purchased as 99.9% chlorides (except gadolinium which was prchased as the nitrate salt) from Alfa Inorganics. Calcium chloride (ultrapure) was also obtained from Alfa. All other compounds were reagent grade and used as obtained. Stock solutions of the metal ions were made about 5 mM with deionized distilled water. Stock solutions of the chelating agents were made to 0.01 M with either deionized distilled water or methanol. Procedure. Relative emission intensity measurements of the lanthanide complexes were made by adding 1 mL of the stock solution of the chelating agent and 1mL of the stock lanthanide solution to a 1 cm square quartz cell. The relative emission intensity was recorded for each chelating agent at room temperature. To evaluate the influence of pH on the Tb(II1)-DPA complex, 1mL of 9.30 mM DPA solution and 1 mL of 0.190 mM Tb(II1) solution were placed in the cell. After the initial pH and relative intensity were measured, small aliquots of 6 M NaOH or HC1 were added to the cell. The pH and relative intensity were measured after each addition. This procedure was repeated until the range of 1.1to 12.0 was covered. All subsequent studies were conducted in the optimum pH range; all solutions were made at a pH of 6.5 with 0.01 M piperazine buffer.
0003-2700/82/0354-2022$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
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Table I. Relative Terbium Luminescence for Various Chelating Agents chelating agent
solventa
excitation wavelength, nm
dipicolinic acid (DPA) phenylmalonic acid diphenylacetic acid pyridine- 2,5-dicarborylic acid phthalic acid 0-benzoylbenzoic acid diphenic acid lasalocid A pyridine- 2,3-dicarboxylic acid pyridine-9,5-dicarboxylic acid pyridine- 2,4-dicarboxylic acid p-phenyl enebis(methylenedithioacetic acid)
water water methanol water methanol water methanol methanol water water water methanol
312 255 274 360 310 360 358 361 355 299 34 7 3 54
a Chelating agents in water have 0.01 M piperazine buffer with pH 6.5. as 100. Excitation a t A ~ ~ .
Two milliliters of 0.635 mM Tb(II1) solution was titrated in the fluorescence cell with microliter aliquots of DPA (4 mM). The solution was mixed and the relative intensity recorded after each addition. Both solutions contained 0.01 M piperazine buffer at 6.5 pH. To study the effect of a second lanthanide or alkaline earth ion on the Tb(II1)-DPA luminescence, we prepared a terbium reagent. The ratio of DPA to Tb(II1) is 31 for the terbium reagent and the solution is buffered at 6.5 pH with 0.01 M piperazine. Two milliliters of a terbium reagent (terbium 0.318 mM) was added to the fluorescence cell and titrated with microliter aliquots of metal ion stock solutions. Unknown solutions were determined by constructing analytical curves. Calibration curves consisted of six or seven concentration points done in duplicate. Samples were either mixed with DPA and buffer and diluted to volume in class A volumetric glassware or a given volume of the' namplelstandard was mixed with an equal volume of DPA solution that was buffered at pH 6.5.
re1 intens at 545 nm 100
84.5 40.4 38.6 15.7 13.1 9.46 9.27 3.04 2.28 1.61 1.48
Relative intensity of Tb-DPA arbitrarily selected
0
8 230
,
...,..,
270
310
WAVELENGTH, NM
Figure 1. The absorption spectra of a terbium reagent (-) and DPA of the same concentration as in the terbium reagent (-). Spectra were recorded at room temperature at pH 6.5.
RESULTS AND DISCUSSION By applying information gained through the use of lanthanide ions as probes to investigate the metal binding sites of metalloproteins (13--15),we have developed a spectrophotometric method to determine lanthanide and alkaline earth ions which is based on nonradiative intramolecular energy transfer. A number of chelating agents were investigated with Tb(II1) as the metal ion, but the strongest emission was observed for the Tb(dipico1inate) complex. Several characteristics were used in the ielcreening of perspective sensitizers. The chelating agent must bind to the lanthanide ions strongly. The ligand should be water soluble or soluble in a solvent that is miscible with water to avoid an extraction (16),should insulate the cation from quenching by water, must contain at least one chromophore with a lhigh molar absorptivity to absorb the incident radiation, and must be able effectively to transfer energy to the cation. The relative terbium luminescence for the chelating agents is given in Table I. The exciting monochromator was varied to achieve the maximum emission at 545 nm. The Tb(111)-DPA complex exhibited the strongest luminescence, and it was selected as the candidate for further study. The absorption spectra of a terbium reagent (DPA to Tb(111) ratio 3:l; Tb(II1) 0.318mM) diluted by a factor of 9/100 in buffer solution at pH 6.5 is shown in Figure 1. The spectrum of DPA without terbium but of the same concentration is also illustrated in Figure 1. More of the carbonyl fine structure is visible i n the spectrum of the terbium reagent. The piperazine buffer has no significant absorption above 200 nm. Terbium europium, and dysprosium are the only lanthanide ions that show detectable luminescence in DPA solutions. The excitation and emission1spectra for a terbium reagent (Tb(II1) 0.318 mM) are presenttrld in Figure 2. The excitation spectra
200
300
400
500
600
700
WAVELENGTH,NM Flgure 2. Luminescence spectra of a terbium reagent. Excitation spectrum recorded with an emission wavelength of 545 nm and the emission spectrum recorded with an excitation wavelength of 250 nm. Both spectra were recorded at room temperature with 1 mm (5.5 nm band-pass) excitation and 0.5-mm (2.75 nm band-pass) slits, and the solution contained 0.01 M piperazine buffer at pH 6.5.
for europium and dysprosium DPA complexes under identical experimental conditions have essentially the same shape as the one shown for terbium; however, the valley at 270 nm is not as deep for dysprosium. The relative intensity at 614 nm (Amm) for the europium-DPA complex excited at 250 nm is approximately as intense as the Tb(II1) emission at 545 nm.
2024
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
o ~ o o 0
0
0 0
0
0
0
0
0 0 0
0
0
0 0
0 0 0
0" 0 0
1
3
5
7
9
DPA/TERBIUM Flgure 3. The luminescence titration of Tb(II1) with DPA. Two millillters of Tb(II1) (6.35 X M in 0.01 M piperazlne buffer at pH 6.5) was titrated in the fluorescence cell with microliter allquots of 4 mM
DPA.
The relative intensity at 477 nm (k-) for dysprosium is about one-tenth the value found for Eu(II1) and Tb(II1) when it is excited a t 250 nm. The piperazine buffer has no detectable luminescence but DPA has a very wide, weak emission band (Amax: excitation, 245 nm; emission, 400 nm). The luminescence intensity of the lanthanide ions in DPA solution depends upon the wavelength of the exciting radiation, the DPA concentration, the temperature, and the solution pH. Figure 3 shows the effect of the DPA concentration on Tb(II1) emission intensity at 545 nm when excited at 250 nm. The maximum luminescence occurs at a 3:l ratio of DPA to Tb(III), as expected for a Tris-DPA complex. Lanthanide complexes of DPA have been well characterized in solution by formation constants (17) and proton magnetic resonance (18) and in the solid state by X-ray diffraction (19). The reduction in the observed intensity after a 3:l ratio is due to an inner filter effect. Although the relative intensity continues to decrease after a DPA to Tb(II1) ratio of 9, the curve levels off and the rate of change decreases. The position of the excitation spectrum also shifts with changes in concentration. In Figure 2 maximum intensities appear a t 250 and 290 nm in the excitation spectrum. However, when the solution is diluted 9 to 100 with buffer, these peaks are at 225 and 272 nm. There is also a shift during the titration shown in Figure 3. At a DPA to Tb(II1) ratio of 0.26:1, the peaks are 226 and 273 nm but quickly shift to the values presented in Figure 2 as the titration progresses. At very high DPA and Tb(II1) concentrations the peaks are shifted to longer wavelengths (see Table I). These wavelength shifts represent the best compromise between maximum energy transfer and minimum absorption by DPA molecules not bound to Tb(II1) ions. The curve in Figure 3 clearly illustrates that when DPA is used as a spectrofluorometric reagent, the concentration of DPA must be the same in both the sample and standard. Also it is prudent to employ a large excess of DPA so that the determination is conducted in a region on the curve where changes in relative intensity are small even though there is loss in sensitivity. The effect of pH on the terbium luminescence has been described previously (20, 21) and our results agree with the literature values. A simple, one-step fluorometric method for the determination of nanomolar concentrations of terbium using DPA was developed by Sherry (20). We have applied this method to the direct determination of Tb, Eu, and Dy. When 2 mM DPA is used and when the samples are excited at 250 nm, analytical calibration curves are linear (correlation coefficient 0.999) over a wide range: 50 mM to 5 nM for T b and Eu, and 50 mM to 50 nM for Dy. The determination of calcium and lanthanide elements other than Tb, Eu, and Dy must be done indirectly and requires the use of a terbium reagent. When the metal ions are added to the terbium reagent, the luminescence at 545 nm
METAl ION CONC x
lo5
Flgure 4. Relative intensity for a terbium reagent (Tb(DPA)z-0.314
mM in 0.01M piperazine at pH 6.5) upon addition of Ca(I1)and trivalent lanthanide ions: (A) Ca(I1)(A),Dy(II1) (0),Yb(II1) (O),Er(II1) (V), Ho(II1)(X), and Eu(II1) (A);(B) Sm(II1)(X), Pr(II1) (O),Lu(II1) (O), Gd(II1) (A),and Nd(II1) (A).
Table 11. The Logarithms of the Formation Constants for DPA Complexes metal ion Ca( 11) Co(11) CU(I1) DY(111) Eu(111) Gd(111) a Values from ref
log K ,
metal ion
4.05
Mg(W Mn(11) Ni(I1) Tb(111) Zn(11)
7.10 9.14' 8.69' 8.84' 8.74a
log K , 1.66
4.72 7.45 8.6ga 7.29
22, other values from ref 23.
is reduced for all metals except magnesium. Magnesium with its smaller ionic radius does not appear to compete with terbium for the DPA ligand. The initial reduction is about the same for all of the lanthanide ions as shown in Figure 4. Ca(I1) also decreases the relative intensity but not as much as the lanthanide ions. Variation among the lanthanide ions emanates from differences in formation constants (22) and experimental error. The reduction in relative emission intensity that is observed in Figure 4 is not luminescence quenching in the classical sense but occurs because new nonluminescent DPA complexes are formed. These new complexes are formed by removing DPA from the terbium reagent. The luminescence decreases since the number of DPA sensitizers bound by Tb(II1) ions is decreasing. Thus, the indicrect method is based on equilibria between Tb-DPA complexes and the added metal ions, and the results are predictable from the formation constant values given in Table 11. Lanthanide ion values are similar while the value for
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
Table 111. Determination of Metal Ions by Spectrofluorometric Analysis with Dipicolinic Acid sample no.
metal ion
concentration, wg/mL actual measda
% error
Tb Eu DY
3.23 3.27 +1.2 1.39 -1.4 1.41 1.71 -1.7 1.74 Hob 1.61 -1.8 1.64 2 Cab 0.505 0.501 -0.8 ND 0.588 Mg 3d Tb + 2.5 2.07 2.02 Nd 0 0.538 0.538 4d Tb 2.06 +2.0 2.02 Nd 0.268 Ho 0.257 Nd, Ho b , e 0.525 0.544 t 3.6 gf Tb 3.21 -0.6 3.23 a Reported values are the average of at least duplicate analysis. Analyzed by the indirect method. Not determined. Equal volumes of sample/standard and DPA with buffer added directly to fluorescence cell. e Nd( 111)used as the standard. Microcell containing 5 p L of solution used in this determination. 1
calcium is 4 orders of magnitude lower than the one for terbium. Magnesium does not affect the terbium emission since its formation constaint for DPA is too small to permit it to remove DPA from terbium complexes. Transition metal ions also form strong metal complexes and could be determined by using the terbium reagent. However, the presence of transition metal ions will interfere with the fluorometric method outlined here. With the initial linear portions of the curves shown in Figure 4 as analytical calibration curves, all the lanthanide ions and calcium can be determined in dilute aqueous solution. When the added metal ion (concentration is equal to the original Tb(II1) concentration (0.318 mM or 311.8 X M as shown in Figure 4) the reduction in Tb(II1) luminescence is about 70% for the lanthanide ions and about 10% for calcium. The reduction is about 10% for the lanthanide ions when the concentration of the added metal ion is about one-tenth the terbium concentration (at about 3 X M in Figure 4). Consequently, one could use a dilute terbium reagent ( M) and establish a detection limit of about lo4 M or 0.1 pg/L for the metal ions using the indirect quenching curve. The Dy(II1) detection limit is an order of magnitude lower when it is determined by the indirect method. Samples containing lanthanide or alkaline earth ions were determined by the spectrofluorometric method, with the results being tabulated in Table 111. The experimental strategy of this spectrofluoro~metricmethod involves the direct determination of Tb(III[),Eu(III), and Dy(II1) and then an indirect determination of other ions using a terbium reagent. This study has concentrated on the use of a terbium reagent. However, a europium or dysprosium reagent would serve the same purpose. Experimentally, Tb(III), Eu(III), and Dy(II1) are evaluated directly by adding a constant amount of DPA with buffer to a series of standard solutions and the sample(s). Then, DPA and buffer are added to the standard and sample solutions so that the DPA to Tb(II1) concentration ratio is 3:l. This second serim of solutions allows the other ions to be determined. If no terbium is present in the sample or if the terbium concentration is less than the other ions, the sample is added to a terbium reagent. Samples 1 and 2 are in the middle of the linear analytical concentration range (see Table 111)and were determined with an error of less than 2 % .
2025
For even faster and easier analysis, the sample/standard and spectrofluorometric reagent may be mixed in the fluorescence cell with only a slight loss of accuracy (see the results for samples 3 and 4 in Table 111). Simultaneous determination of four ions is only possible when Dy(III), Tb(III), and Eu(II1) are present as in sample 1. In this sample Dy(III), Tb(III), and Eu(II1) are determined directly. Then, the total concentrations of Dy(III), Eu(III), and Ho(1II) are found indirectly, which yields the Ho(II1) concentration by difference. If two or more nonluminescing ions are present in the sample, the total concentration is estimated as in sample 4 where the total Ho(II1) plus Nd(II1) concentration is based on a Nd(II1) standard. We selected two of the most divergent examples from Figure 4; however, the relative error increased only slightly since the initial section of all the curves was quite similar. Finally, when only very small samples are available, the method is readily adaptable to microsampling. Fivemicroliter volumes were used for sample 5. These samples and standards were prepared with volumetric glassware but 3 p L of the DPA reagent could be mixed with 3 I~.L of a sample or standard in a plastic microweighing dish. A 15.2-ng quantity of Tb(II1) was determined in sample 5 with less than 1% error. Relative emission intensity for the microcell is about one-tenth the value observed in a standard 1-cm cell so the detection limit is about 10 pg/L or 50 pg for a 5-pL sample in the microcell. In conclusion nonradiative intramolecular energy transfer provides a quick, easy, and reliable method to determine calcium and lanthanide samples. Since magnesium ions do not reduce the terbium luminescence, calcium can be determined in dilute aqueous solution in the presence of magnesium. This feature holds great promise for biological and environmental samples where these two ions are always found together.
LITERATURE CITED
N
(19) (20) (21) (22) (23)
Luk, C. K. Biochemistry 1971, IO, 2838-2843. Wolfson, J. M.; Kearns, D. R. Biochemistry 1975, 14, 1436-1444. Martin, R. B.; Rlchardson, F. S. 0.Rev. Biophys. 1979, 12, 181-209. Eislnger, J.; Lamola, A. A. Biochim. Siophys. Acta 1971, 2 4 0 , 299-312. Fassei, V. A. Anal. Chem. 1960, 32,19A. Sarvin, S.B. Zavod. Lab. 1963, 2 9 , 131-139. Onishl, H.; Banks, C. V. Talanta 1963, IO, 399-406. Brittain, H. G. Anal. Chem. 1977, 4 9 , 969-972. Van Loon, J. C.; Galbraith, J. H.; Aarden Analyst (London) 1971, 9 6 , 47-50. Dleke, G. H.; Hall, L. A. J . Chem. Phys. 1957, 2 7 , 465-487. McCarthy, W. J.; Winefordner, J. D. Anal. Chem. 1966, 38,848-853. Alberti, G.; Massuccl, M. A. Anal. Chem. 1966, 38,214-216. Mliler, T. L.; Nelson, D. J.; Brlttain, H. G.; Richardson, F. S.; Martin, R. B. FEBS Lett. 1975, 58,262-264. Nelson, D. 1.; Miller, T. L.; Martin, R. B. Bioinorg. Chem. 1977, 7 , 325-334. Miller, T. L.; Cook, R. M.; Nelson, D. J.; Theoharides, A. D. J . Mol. Biol. 1980, 141, 223-226. Fisher, R. P.; Winefordner, J. D. Anal. Chem, 1971, 43, 454-455. Grenthe, I. Acta Chem. Scand. 1963, 17, 2487-2498. Donato, H., Jr.; Martln, R. B. J . Am. Chem. SOC. 1972, 9 4 , 4129-413 1, Albertsson, J. Acta Chem. Scand. 1972, 2 6 , 985-1004. Barela, T. D.; Sherry, A. D. Anal. Biochem. 1976, 7 1 , 351-357. Brittain, H. G. Jnorg. Chem. 1978, 17, 2762-2766. Grenthe, I. J. Am. Chem. SOC. 1961, 83,360-364. Chung, L.; Rajan, K. S.;Merdlnger, E.;Grecz, N. Blophys. J . 1971, 1 1 , 469-482.
RECEIVED for review April 15, 1982. Accepted July 19,1982. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for the support of this research and to the National Science Foundation for providing an instrumentation grant to purchase the Aminoco-Bowman spectrofluorometer (CDP-7923794).
