Anal. Chem. 1987, 59, 1122-1125
1122
Submicrogram Determination of Lanthanides through Quenching of Calcein Blue Fluorescence Harry G . Brittain The Squibb Institute for Medical Research, P.O. Box 191, New Brunswick, New Jersey 08903
I t has been found that trace levels of lanthanide ions efficiently quench the fluorescence of calceln blue and that an analytical method based on thls quenchlng Is far more sensitive (0.01-0.02 pg/mL depending on the ldentliy of the lanthanide Ion) than exlstlng methods based on absorption spectrophotometry. The sensltlvlty levels are comparable to those noted for fluorescence observatlon of the few lanthanides which luminesce dlrectly, but the calcein blue method may be applied equally we# to any lanthanide Ion. Interference by dlvalent transltlorrmetal Ions has been noted, but the lower degrees of quenching efficlency by these ions ensure that slgnlflcant Interference will exlst only at relatively high metal levels.
A large number of reagents have been proposed for the spectrophotometric determination of individual lanthanide ions, or for the total lanthanide content (1). The sensitivity limit for these techniques is around the pg/mL level. More sensitivity can be obtained by using fluorometric methods ( Z ) , but only Sm(III), Eu(III), Tb(III), and Dy(II1) complexes are sufficiently emissive for the development of analytically useful methods. The determination of other lanthanide ions below the pg/mL level requires the use of atomic spectroscopic methods, such as inductively coupled plasma atomic emission spectrometry ( 3 ) . The use of fluorescence quenching methods for the determination of lanthanide ions has received insufficient attention, although two chelating dyes derived from methylumbelliferone could be useful for this purpose. Calcein blue (abbreviated henceforth as CB)
has been used to develop sensitive methods for the determination of Ni and Cr ( 4 ) ;Ca, Mg, and Fe (5); Ga (6); and Zr (7). Far less work has been carried out with the structurally related dye, methyl calcein blue (abbreviated as MCB) CH3
I
although MCB has been shown to be useful in the chelometric determination of AI(III), Ni(II), and Mn(I1) (8). For reasons which will become apparent in the following sections, 0003-2700/87/0359-1122$01 SO/O
fluorescence quenching studies were also performed on the parent dye itself, 4-methylumbelliferone (abbreviated as UMB)
In the present work, it will be shown that quenching of calcein blue fluorescence by lanthanide ions can be used to develop extremely sensitive assays for individual or total lanthanide content and that the method is useful for all members of the lanthanide series. Quenching of methyl calcein blue or methylumbelliferone fluorescence is far less effective, but important to the data interpretation. Quenching by divalent transition-metal ions can also be observed, indicating that these ions are capable of interfering with the determination of lanthanide concentrations.
EXPERIMENTAL DETAILS Reagents. All materials used during the course of the present work are commercially available and were used as received. Calcein Blue Reagent. Coweigh 0.010 g of calcein blue dye and 1.25 g of tris(hydroxymethy1)aminomethane (THAM buffer). These materials are dissolved in 75 mL of water, the pH adjusted to 8.0 with 3 M HC1, and diluted quantitatively to 100 mL with water. It should be noted that the calcein blue reagent solution undergoes slow decomposition after preparation. For best results, the solution should be prepared on a weekly basis and stored at reduced temperatures when not being used. The concentration of CB in the reagent is therefore 0.30 mM, and the concentration of THAM is 0.10 mM. Methyl Calcein Blue and Methyumbelliferone Reagents. These reagent solutions were prepared in a manner exactly identical with that just described for calcein blue. Metal Zon Standard Solutions. Stock solutions of lanthanide and transition-metal ions were made at suitable levels to establish calibration curves. For the lanthanides, 5.0 mg of hydrated lanthanide nitrate was weighed, dissolved in 40 mL of water, and diluted to 50 mL in a volumetric flask. The exact concentration of these stock solutions depended on the particular lanthanide used but was about 0.25 mM (100wg/mL). Exact concentrations were established through EDTA complexometric titrations. Due to their lower quenching efficiencies, more concentrated stock solutions of transition-metal ions were required. Sufficient quantities of the nitrate salts were weighed and diluted to yield 2.5 mM solutions. For the MCB and UMB work, concentrations of metal ions up to 25 mM were required. Apparatus. All data were obtained on a Spex Fluorolog instrument, with the DMlB Spectroscopy Laboratory Coordinator being used to control the data acquisition and processing. Calcein blue, methyl calcein blue, and methylumbelliferone can all be excited at 370 nm, with the emission maximum being located at 435 nm. One can simply read the emission intensity (in the conventional arbitrary units) directly. Alternatively,one can scan the CB fluorescence spectrum between 380 and 600 nm and use the integrated band area as the intensity measurement. Procedure. Pipet 3.0 mL of the calcein blue (or methyl calcein blue) reagent into a fluorescence cuvette and obtain the 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
48
I 10
L
0
#
/
0
B
I
1123
/
I
#'
6
250
I
I
I
I
1
300
350
400
450
500
WAMLENtTH
550
(NM)
Figurs 1. Excitation (solid trace) and fluorescence (dashed trace) spectra of calcein blue, buffered at pH 8.0. The excitation maximum is located at 370 nm, while the emission maximum is found at 435 nm.
fluorescence intensity of this solution. Pipet 10 pL of the lanthanide ion solution into the cuvette, and rescan the fluorescence spectrum. The addition should be repeated in 10-pL incrementa until a total of 100 pL of the lanthanide solution has been added. The intensity of fluorescence at 435 nm should be measured after each addition. For exceedingly low levels of free Ln(III), titration L be used so that significant increments of either 20 or 30 ~ Lshould degrees of CB quenching can be measured. However, at least 10 additions of lanthanide solution should be added to ensure adequate accuracy in the Stern-Volmer quenching kinetics. The metal ion concentration can be determined by two methods. A calibration curve relating observed emission intensities with known levels of metal ion may be constructed and concentrations obtained after regression analysis is used to evaluate the curve parameters. Alternatively, one can use a Stern-Volmer kinetic analysis (which will be described in a following section), since a direct link between metal ion concentration and fluorescence quenching is an integral part of this analysis.
RESULTS AND DISCUSSION The ionized forms of calcein blue and methyl calcein blue are highly fluorescent upon UV excitation, and the observed spectra are characteristic of the methylumbelliferone fluorophor. The excitation spectrum features a maximum at 370 nm, while the emission spectrum peaks at 435 nm. These two spectral types have been illustrated in Figure 1, where one notes miminal overlap between the excitation and emission spectra. The lack of an observable 0-0 band and the complete mirror image of the two spectra indicates that the compounds do not undergo any photochemistry or molecular rearrangement in the respective excited states. Addition of any lanthanide ion (noted generically as Ln) to a CB solution buffered a t pH 8 results in an extremely efficient quenching of the CB fluorescence. The CB compound contains iminodiacetate groups (ionized at pH 8), and these are known to bind lanthanide ions with substantial binding constants (9). It may therefore be concluded that the high efficiency of the quenching mechanism is due to complexation of the lanthanide quencher with the CD donor. This mechanism is normally referred to as "static" quenching. The effect of lanthanide ion addition on CB fluorescence intensity is illustrated in Figure 2, using Gd(II1) as the example. It is apparent in Figure 2 that two distinct quenching regions exist, and this behavior was observed for all members of the lanthanide series. The quenching is extremely efficient at low concentrations of Ln(II1) and becomes significantly less efficient at higher concentrations. Since the dye concentration used was 0.3 mM, it is reasonable to interpret the quenching data in terms of two competing equilibria
+ CB + Ln(CB) Ln + 2CB e Ln(CB)? Ln
(1)
Equation 2 would dominate at low ratios of Ln/CB, while eq 1would dominate as the concentration of Ln and CB became comparable. The very inefficient quenching observed at the
c O
I
2
I
4
[Gd3+]
I
1
I
6
B
10
x lo'
Flgure 2. Quenching of caicein blue fluorescence by lanthanide ions, as illustrated by Gd(II1). The solid curve depicts the CB emission
intensity as a function of added Gd(II1) and is given in arbitrary units (left-hand scale). The dashed curve depicts the quenching as computed through the Stern-Volmer equation (right-handscale). The portion of the curve most suitable for analytical work corresponds to Gd(II1) concentrations less than 10 pM. highest Ln(II1) concentrations represents outer-sphere quenching of residual CB fluorescence by free Ln(II1). The efficiencies observed in the lanthanide ion quenching of MCB fluorescence were observed to be far less than those observed with CB. No division of concentration ranges was observed, and the calibration curves smoothly increased until a limiting value for the quenching was reached. It is wellknown that lanthanide ions have a poor affinity for amino acids below pH 9 (9),and consequently any quenching would reflect contributions from collisional encounters as well as the static mechanism. This result is seen as confirming that the extremely efficient quenching noted for the CB system is related to the formation of a complex species. Even lower quenching efficiencies were observed with the UMB system. The low efficiencies observed with the MCB and UMB systems indicate that these materials are not useful for analytical determinations. The low concentration region of the CB system contains a response of analytical interest, and consequently the lanthanide quenching of calcein blue was investigated in more detail. As may be noted in Figure 2, the CB fluorescence is quenched by Ln(II1) in a nonlinear fashion. When the data are processed through the Stern-Volmer equation
(3) a linear relation was obtained over the lowest Ln(II1) concentration range. In eq 3, Io is the intensity of CB fluorescence before any Ln addition, Ii is the CB fluorescence intensity after the ith addition of Ln, K,, is the Stern-Volmer quenching constant, and [Q]is the concentration of Ln(II1) quencher in the sample. Since the concentration of Ln is known, the quenching data are used to obtain K,. The plot of ( I , - Ii)/Ii against the concentration of Ln quencher yields K,, as the slope of the resulting line and an intercept of zero. The Stern-Volmer constants corresponding to the lanthanide ion quenching of calcein blue, methyl calcein blue, and methylumbelliferone fluorescence are found in Table I. It is apparent that the CB quenching efficiencies average approximately 200X the corresponding MCB quenching efficiencies and that the MCB and UMB quenching efficiencies are comparable. The very large magnitudes associated with the SternVolmer constants permit the detection of extremely low lanthanide ion concentrations. A 1% emission intensity reduction is easily measurable with digital photon counting, and such quenching would amount to an (Io- n / I value of 0.01. For
1124
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
Table I. Stern-Volmer Constants Obtained from t h e Quenching of Calcein Blue, Methyl Calcein Blue, and 4-Methylumbelliferone Fluorescence by Trivalent Lanthanide-Metal Ions“ I,nt I11 I
calceiti blue
La Ce
66 600 78 600 95 800 98 900 101000 114 000 111000 115000 117 000 120000 113000 109 000 9’; 900 90 500
Pr Sd St11
Eu Gd Tb D?. Ho Er Tm I’b Lu
methyl calcein blue
4-methylumbelliferone
336 39: 184
24 1 299 381 105 131 163 162 163
508 540 574
576 581 550 585 579 554 194 178
1-1 151
126 361 315
Table 11. Apparent Association Constants of t h e Complexes Formed by Calcein Blue a n d Methyl Calcein Blue with Trivalent Lanthanide-Metal Ions calcein blue
La Ce Pr Nd Sm Eu Gd Tb DY Ho Er Tm Yb Lu
66 360 78 300 95 410 98 500 100 600 113 500 113500 114500 116500 119 500 112500 108 600 97 540 90 160
TM(I1)
calcein blue
methyl calcein blue
4-methylumbelliferone
Mn Fe co Ni cu Zn
28 900 21 100 24 700 22 300 27 900 13200
2 190 4 300 9 020 12 600 19 500 5 170
502 548 640 621 585 515
“Each Stern-Volmer constant has the units of L/mol, and is associated with an approximate 5% error.
163
“Each Stern-Volmer constant has the units of L ’mol and is associated with an approximate 5% error.
Ln(II1)
Table 111. Stern-Volmer Constants Obtained from the Quenching of Calcein Blue, Methyl Calcein Blue, and 4-Methylumbelliferone Fluorescence by Divalent Transition-Metal Ionsa
methyl calcein blue 95 98 100 103 109 111
114 117 119 122 125 128 130 133
Gd(III), this degree of quenching would correspond roughly to a 0.1 pM lanthanide ion concentration, or 0.015 pg/mL. This level of quantitation compares favorably with the 0.01 pg/mL limit associated with atomic absorption methods. The detection limits associated with inductively coupled plasma spectroscopy are an order of magnitude lower, but this instrumentation is considerably more expensive to implement. The quantitation limit associated with the calcein blue method is comparable to the fluorescence methods known for Sm, Eu, Tb, or Dy, but the current method is superior in that it may be applied with equal sensitivity to all members of the lanthanide series. Since it may be assumed that the fluorescence quenching contains contributions from both collisional and complexational phenomena, the Stern-Volmer constants of Table I can be used to estimate the complexational binding constants. For this situation, the Stern-Volmer equation may be written as where Kq is the Stern-Volmer constant for collisional quenching and K , is the association constant for the donorquencher pair (IO, 11). Since methylumbelliferone does not possess any sites capable of chelating a lanthanide ion, the Stern-Volmer quenching constants obtained for Ln(II1) quenching of UMB may be taken as the pure collisional contribution, Kq. With this assumption, the Stern-Volmer constants of Table I were used to compute the apparent as-
Table IV. Effect of Added Cu(I1) on t h e Quenching of Calcein Blue Fluorescence by Gd(II1) actual [Gd(III)I, PM
(Io - 011”
(I, - 0 / I b
1.00 2.00 3.00 4.00 5.00
0.114 0.227 0.341 0.454 0.568
0.114 0.228 0.342 0.456 0.570
(Io -
n/rc
0.115 0.230 0.345 0.460 0.575
” Stern-Volmer quenching parameter obtained for the stock Gd(111) solution. Stern-Volmer quenching parameter obtained for the stock Gd(II1) solution to which 2.0% Cu(I1) had been added. Stern-Volmer quenching parameter obtained for the stock Gd(111) solution to which 5.0% Cu(I1) had been added. sociation constants of Table 11. The calcein blue association constants average approximately lOOX smaller than the corresponding constants known for 1:l iminodiacetate complexes (9). This difference is certainly due to the extra steric bulk of the methylumbelliferone fluorophor associated with the CB reagent. The methyl calcein blue association constants are observed to be considerably smaller but are exactly within the range anticipated for lanthanide amino acid complexes (9). Possible interference by divalent transition-metal ions with the lanthanide ion determination was also investigated. It was found that all common transition-metal ions were capable of quenching the fluorescence of CB, MCB, and UMB, although they did so at significantly different efficiencies than did the lanthanide ions. As shown in Table 111, the transition-metal ion quenching of CB fluorescence is only one-fifth as effective as was observed with the lanthanide ions. At the same time, the quenching of MCB fluorescence was found to be considerably more efficient than with the lanthanides and exhibited large variations in magnitude. The UMB quenching efficienciesassociated with the divalent transition metals were almost comparable to the lanthanide quenching efficiencies and were found to exhibit considerably less variation. Based on the CB Stern-Volmer quenching constants, a 0.25 pg/mL detection limit was calculated. This limit is an order of magnitude higher than that calculated for the lanthanide ions, indicating that significant interference would only be noted when the concentration of transition-metal ion approached 10% that of the lanthanide ion. To test this latter hypothesis, the effect of Cu(I1) interference on the determination of Gd(II1) was investigated in detail. This particular ion was chosen since it represents a real possibility for interference and is also one of the stronger transition metal ion quenchers of calcein blue fluorescence. A 0.300 mM Gd(II1) stock solution was prepared and used to titrate the calcein blue reagent. Two other Gd(II1) stock solutions were prepared (each 0.300 mM in Gd); one with 2%
1125
Anal. Chem. 1087, 5 9 , 1125-1129
Table V. Apparent Association Constants of the Complexell Formed by Calcein Blue and Methyl Calcein Blue with Divalent Transition-Metal Ions methyl Ln(II1)
calcein blue
calcein blue
Mn
28 400 20 550 24 060 21 680 27 320 12 685
1690 3 750 a 380 11980 18 920 4 650
Fe co
Ni CU Zn
Cu(I1) and the other with 5% Cu(I1). The response of the calcein blue reagent to these solutions was obtained, and the Stern-Volmer quenching parameters thus measured are collected in Table IV. It was concluded that the addition of 2% Cu(I1) yielded only a 0.4% error in computed Gd(II1) concentrations, while the addition of 5% Cu(I1) yielded only a 1.3% error. Apparent association constants for the transition-metal complexes with calcein blue and methyl calcein blue were computed from the quenching data by using the same analysis
as previously described. These constants are found in Table V, and comparison with analogous iminodiacetate and amino acid complexes (9) indicates that the dye complexes are less stable than those of the parent chelating group. This observation implies that the steric bulk of the umbelliferone group interferes with the complexation process, as had been inferred for the lanthanide-dye complexes.
LITERATURE CITED Howell, J. A.; Hargis, L. G. Anal. Chem. 1988, 58, 108. Wehry. E. L. Anal. Chem. 1988, 58 13. Yoshida, K.; Haraguchl, H. Anal. Chem. 1904, 5 6 , 2580. Wllklns, D. H. Talanta 1960, 4 , 182. Escarrllla, A. M. Talanta 1988, 73,363. Elsheimer, H. N . Talanta 1867, 14, 97. Hems, R. V.; Klrkbrlght, G. F.; West, T. S. Anal. Chem. 1970, 4 2 , 784. Wilkins, D. H. Talanta 1980, 23, 309. Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum: New York, 1974, Vol. 1. Boaz, H.; Rollefson, G. K. J. Am. Chern. Soc. 1950, 72, 3435. Keizer, J. J . Am. Chem. Soc. 1983, 705,1494.
RECEIVED for review September 2, 1986. Accepted January 9, 1987.
Development of Sensitive Multiwavelength Fluorescence Detector System for High-Performance Liquid Chromatography Kiyoshi Tanabe,' Mark Glick, Benjamin Smith, Edward Voigtman, and James D. Winefordner*
Department of Chemistry, University of Florida, Gainesville, Florida 32611
A sensitive, multiwavelength fluorescence detector system for hlgh-performance llquld chromatography (HPLC), which slmuttaneously monitors four fluorescence wavelengths on four Interference fllter-photmuttlpller tube detector channels, has been developed to reallre both hlgh sensltlvlty and hlgh selectlvlty. Performance of the system has been estimated by uslng several polycycllc aromatlc hydrocarbons ( PAHs). Detectlon llmlts In the picogram range and llnearlty of callbratlon curves over 104-106 were obtained for most PAHs. Repoduclbillty was 0.7-5.9 % (relatlve standard devlatlon, N = 7) and stablttty was
