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Fluorescence of magnesium-, calcium-, and zinc-8-quinolinol complexes
ANALYTICAL
BIOCHRMISTRY
Fluorescence
5, 345-359
(1963)
of Magnesium-,
Calcium-,
quinolinol SHIZUO From
the
WATANABE,2 DONNA
Cardiovascular Medical
Zinc-8-
Complexes1 WILLIAM TROTTIER
Research Center, San Received
and
Institute, Francisco, July
FRANTZ,3 University California
of
AND
California
16, 1962
INTRODUCTION
About ten years ago, R. N&&en (l-3) made a series of extensive studies on the binding (or stability) constants of metal chelates of 8quinolinol, using the light absorption method, the potentiometric method, and also the method of solubility equilibrium. For example, the pk’ values at zero ionic strength and 20°C were reported to be 4.74, 3.27, and 8.56 for Mg”+, Ca?+, and Zn”+ binding, respectively (3). In 1959, D. Schacter (4) reported that an ethanol solution of magnesium-8-quinolinol exhibited fluorescence with a peak intensity at 530 ml*. He showed that this fluorescence was dependent on pH and specific for Mg’+. Among eleven other divalent cations he tested, Zn2+ was the only one that exhibited a significant intensity of fluorescence, i.e., approximately onehalf that observed with Mg?+. Ca2+ and Sr?+ exhibited negligible fluorescence and the other eight cations did not exhibit fluorescence. By utilizing these fluorescence characteristics, Schacter introduced a sensitive and specific method for magnesium determination in serum and urine. In the present paper, the fluorescence of Mg?+, Ca”+, and Zn’+ chelates of S-quinolinol is studied further in aqueous solutions (buffered with 0.1 M triethanolamine or 0.1 M tris(hydroxy)methylaminomethane) as well as in p-dioxane, which is found to be better than ethanol in increasing the sensitivity of the method; the results are analyzed in terms of the (relative) molar fluorescence and the binding constant. ‘This work was supported by grants from the American Heart Association and U. S. Public Health Service (H6085). ’ Established Investigator of the American Heart Association, Inc. 3The early part of this work was done while W. Frantz was on the Summer Research Fellowship of the American Physiological Society in 1960 at the Dartmouth Medical School, Hanover, New Hampshire. Present address: Department of Physiology, University of Michigan, Ann Arbor, Michigan. 4 Dartmouth Medical School, Hanover, New Hampshire. 345
346
WATANABE,
FRANTZ,
AND
TROTTIER
As for the sensitivity of the fluorescence method for Mg2+ determination, the molar fluorescence of Mg2+ chelate is shown to be 8 x loj (practical units-see definition in the section Materials and Methods) in p-dioxane. Even in aqueous solutions, it is found to be about 7 x IO4 and, accordingly, the sensitivity of this fluorescence method is still far superior to that of the light absorption method [the molar absorbancy = 2 x 103; cf. refs (2), (3), (5)], although the practical fluorescence unit could be compared to an absorbancy unit only in the practical purpose, depending on the commercially available instrument. As to the specificity of this fluorescence method for Mg*+, the molar fluorescence as well as the binding constant are found to make contributions to it. Ca*+-8-quinolinol as well as Zn2+-S-quinolinol is found to exhibit the fluorescence at 530 my upon activation at 360 rnp, but the molar fluorescence for both Ca2+ and Zn2+ chelates is found to be less than one-third of that for Mg2+ chelate. The pK values for metal binding with B-quinolinol obtained by this fluorescence method are 4.4, 3.1, and about 8.1 at an ionic strength of approximately 0.1 and 25°C for MgZ+, Ca2+, and Zn?+, respectively. These values are in good agreement with those reported by N&&en et al. (see above). The analysis that is used in this paper is therefore justified and the fluorescence method for Mg2+ determination gains theoretical basis as firm as the light absorption method. MATERIALS
AND
METHODS
An Aminco-Bowman spectrophotofluorometer equipped with a xenon photomultiplier tube was used to measure the fluorescence. Two slits with 0.5-cm openings were used, one before the activation light reached the 1 X 1 cm sample cell and the other before the emitted light reached the second monochrometer. The fluorescence intensity was expressed by the reading on the per cent transmission scale of the photometer (practical units). Using a mercury lamp as a calibration standard for the light wavelength, the fluorescence peak and the activation peak were observed to be at 530 and 360 rnp., respectively. With Zn?+ chelate, a little deviation in wavelength of the peaks was observed (515 and 375 mp) but all measurements were made of the fluorescence at 530 rnp with activation light of 360 mp. All chemicals used were commercially available and they were used without any further purification. S-Quinolinol and magnesium nitrate (6H,O) were Fisher certified reagents, and calcium chloride, zinc chloride, and magnesium chloride were Baker and Adamson standard purity reagents. Triethanolamine (TEA) 5 and tris (hydroxy) methyllamp and a IP28
s Abbreviations methylaminomethane.
used
are
TEA
for
triethanolamine
and
Tris
for
tris(hydroxy)-
FLUORESCENCE
OF
347
METAL-8-QUINOLINOL
aminomethane (Tris) were Fisher certified reagents; they were used at concentrations of 0.1 M as buffers. Sample solutions were kept in a constant-temperature bath of 25”, except for a few minutes for each measurement of the fluorescence intensity, when an aliquot volume (usually 3 ml) of sample solution was taken to the sample cell compartment which had no temperature control. TREATMENT
OF DATA
In the following discussion Mg”+ or Ca’+ will be denoted by M and undissociated 8-quinolinol by QH. The following equilibria are considered :
QHeQ+H+, M +&S&M,
&i
K
In particular, it is considered that conditions are always such that the equilibrium, M + 2 OH- = M(OH),, can be ignored (see 6). By combining Mass Law expressions, one obtains:
X,‘(Mo - X) (Xo - X) = &&/(KH
+ [H+l)
(1)
Here, the total concentrations of metal ions and 8-quinolinol are denoted by M, and X,,, respectively; the concentration of the complex QM by Xi; K, and K are a dissociation constant and an association constant, respectively, for the equilibria considered above. At constant pH the expression on the right-hand side of Eq. (1 J plays the role of an equilibrium const,wnt which we shall call K’. Differentiating Eq. (1) with respect to M, and combining the result with Eq. (Ii we obtain, at constant pH and X,: aS,‘aM,
= (X, - X)‘/(X/K’
+ (X, - X)/K’
+ (X, - X)?}
(2,
As M, is made very large, S + Xl,, and as M, is macle very small, X+ 0, hence from Eq. (2) it follows that the X(M,) curve will be of a “saturation” type, i.e., at very large M, it will be flat (slope = 0)) and near the origin its slope will be: gmo (aX/aMo)
= X0/( l/K’
+ XO)
(3)
Actually we measure not X, but the fluorescence, f, arising from excitation of QM. If we assume that f = olX (where (Y is the ‘
Equation (4) shows that a plot 01’ the limiting slope, /3, :Ig:Gnst (l/S,, 1
348
WATANABE,
FRANTZ,
AND
TROTTIER
is linear, that the intercept on the /3 axis (l/X, = 0) is l/a, and that the slope of the line divided by this intercept is l/K’. Recalling the definition of K' we may also re-express Eq. (4) as:
P = U/4 + l/bKXo) + t ll(cJ%J 1W&)[H+l Equation (5) shows that at constant X,, p is linear with [H+]. the intercept on the [H+] axis (p = 0) of such a plot, then: (-1~) Accordingly
and :
= &(I
(5) If IIt is
+ KXo)
(6)
: if KXO << 1, then (- IH) = if KXO = 1, then ( -IH) = if KY0 >> 1, then (- IH) =
KB, KH/2, KKHXo
Of course, it is also obvious from the definition of K' that, K' = K, when [H+] = KH, K‘ = K/2, and when [H’l <<&I, >>KH, K’ = K&I PI.
when [H+]
RESULTS
la. Keeping a fixed concentration of Squinolinol (X, = 1.92 x lo-” M) and varying the total concentration of added Mg2+ (n/r,), the intensity of fluorescence (f) at 530 rnp upon activation at 360 rnp was measured at various pH’s (various curves of Fig. 1). The resulting data are closely fitted by theoretical curves (solid lines of Fig. 1) based on the foregoing analysis, and therefore they justify said analysis. In curves such as those in Fig. 1, as M, is indefinitely increased it should be that X * X0 and f + fILlax, then from our assumptions f max = ax,,. In Fig. 2 are shown plots of fmar vs. X, in two different solvents; in such plots the slope is (Y. The data are, in fact, linear plots provided X, < 5 x 1O-5 M. In an aqueous solution (0.1 M triethanolamine buffer, pH 9.45), (Y = 8 X 104; in an ethanolic solution, 80% v/v (Tris buffer, pH 9.5)) a = 33 X 10”. When X, exceeds ca. 5 X 10e5M, the plots bend away from linearity; this is probably due to self-absorption by the increasing concentrations of the complex QM.‘i b. Equation (1) is symmetrical as regards M,, and X,,. Therefore if one starts with a fixed M, and increases X,, or starts with a fixed X,, and increases M,, the fmax a ttained in either case should be the same provided the starting M. equalled the starting X,. However, as shown in Fig. 3 (curves a and b), when we started with a fixed M. (2 X 10e5 M) ‘The fluorescence intensity is linearly with the stance only when the concentration is very low.
roncentration
of fluorescing
WI)-
FLUORESCENCE
349
OF METAL-8-QUINOLINOL
-4 LOG FIG. 1. Fluorescence as a function of Mg” concentration at various pH’s. Wquinolinoll = 1.92 x 10e5M, 0.1 M triethanolamine buffer, and 25”. Solid curves are calculated curves based on the analysis described in the text. Dashed curves are those tracing experimental results.
0
FIG. 2. Maximum intensity of S-quinolinol at 25”. Upper buffer). Lower curve: aqueous
0
4
0
f2
16
of fluorescence observed at various concentrations curve: 80% (v/v) ethanol and pH 9.5 (0.1 IIf Tris solution and pH 9.45 (0.1 M triethanolamine buffer).
350
WATANABE,
FRANTZ,
AND
TROTTIER
and increased X,,’ a lesser fmax was attained and, when the concentration X0 exceeded about 10-3.0 M, the fluorescence intensity decreased with
6
-4
-2
0
LOG [MO OR x0-j FIG. 3. Comparison of fluorescence intensity observed upon addition of increasing concentration of Mg’+ to 2 x lo-‘M &quinolinol with those of Ca2+ and Zn’+ and also with that observed upon addition of increasing concentration of Squinolinol to 2 x IO-“M Mg*+. Curve a: Mg*+ (M,) was added to 2 x lo-’ M 8-quinolinol in 0.1 M triethanolamine buffer, pH 8.95. Curve b: S-quinolinol (X0) was added t,o 2 x lo-‘M Mg’+ in 0.1 M triethanolamine buffer, pH 8.95. Curve c: Ca*+ (MO) was added to 2 x 10“ M 8-quinolinol in 0.1 M triethanolamine buffer, pH 8.95, Curve d: Zn’+ (M,) was added to 2 x 10-‘&f 8-quinohnol in 0.1 M triethanolamine buffer, pH 7.4. Mg” (curve a), Ca*+ (curve c), and Zn’+ (curve d) G M. were added to 2 x 10-‘J14 S-quinolinol in 0.1 M triethanolamine buffer (pH 8.95: curves a and c and pH 7.4: curve d). S-quinolinol (-X0) was added to 2 X lo-’ M Mg” in 0.1 M triethanolamine buffer, pH 8.95.
increasing X,,; this is probably not due to self-absorption of the formed complex QM since the concentration of QM in the presence of 2 X 10e5M magnesium (MO) cannot exceed 5 X 1O-5M, the concentration at which fluorescence of the complex began to show the self-absorption effect (See Fig. 2). This is also not due to the inadequacy of the considered equi‘A solution of metal ion (Me, Cal+, or Zn’+) without S-quinolinol certainly does not exhibit fluorescence but d-quinolinol solution did fluoresce without addition of metal ion. This fluorescence became significant at the concentration of &quinolinol approximately above 10e4 M, and was reduced only partially by addition of EDTA Therefore, this fluorescence is not due to the metal contamination of 8-quinohnol reagent. In curve b of Fig. 3, the difference between the fluorescence intensity with and without addition of magnesium ion was taken as the fluorescence intensity.
FLUORESCENCE
351
OF METAL-8-QUINOLINOL
libria, as will be indicated later in another type of very detailed experiments, but is probably due to absorption of both the activation light and fluorescence light by yellowish color of the S-quinolinol medium which was visually noticeable at these higher concentrations such as 10-“,“M,X or above. c. Bearing these complications in mind, we may estimate K’. For example, for curve a of Fig. 3, X, = 2 X 10-j M and pH 8.95. In this case, the half-maximum fluorescence (fmaxln) is obtained when [Mg*+] = 10-3.67 M. Such a condition is equivalent to setting X = X,/2 in the lefthand side of Eq. (l), i.e., to having K’ = l/Q!!, - X0/2) ; thus from the values just cited, K’ for Mg?+ = 103.G7liter mole-l at pH 8.95. Likewise, K’s for Ca*+ and for Zn2+ may be obtained from curves c and d; K’ for Ca?+ = 102,47 liter mole-l at pH 8.95 and X’ for ZrP+ = 1/(1O-4.s - 1O-5.u) s 1o”,6 liter mole-l at pH 7.4. With Zn2+, however, precipitation took place even when this low pH was used. Although measurements with Zn*+ were done quickly before precipitation became visible, the fact that the plateau had not been reached with curve d reflects the precipitation. The K’ value for Zn”+ cited above was therefore estimated on the assumption that the maximum fluorescence with Zn”+ is the same as that with Ca’+ (2.1 x lO’&). In order to avoid the precipitation with Zn?+, the pH had to be lowered to less than 5. Using 0.1 ill acetic acid-tetramethylammonium hydroxide, the half-maximum fluorescence was observed when [Zn?+] = lo-“.’ M at pH 4.8, indicating K’ for Zn’+ = lO’.l liter mole-’ at this pH. The maximum fluorescence observed at this pH was, however, very small-O.65 X 10’ for 2 X lo+ 1c1 S-quinolinol and accordingly a (Zn”+ chelated = 3.3 x lo3 at pH 4.8. Furthermore, Mg*+, even in :I concentration as high as 0.1 M, did not. produce any fluorescence in the r>reeence of 2 x 10e5M %quinolinol at this pH. Zn”+ chelate was therefore not studied further. d. cr (calculated from the slope of flllax vs. bl,, curves1 and 6’ (calculated from “half-saturation” values as above) were measured in waterethanol and water-p-dioxane solutions, at various pH’s (Fig. 4). The results (see also Fig. 1) can be summarized by saying that K’ is markedly reduced by pH but very little affected by adding p-dioxane; on the other hand, (Y is little affected by pH but markedly increased by p-dioxnnr. The effect of ethanol is qualitatively similar to that of p-dioxane. Ilu,. The method of estimating K’ by locating (fmaxlz) is simple but it does not fit for estimation at lower pH’s where a very high concentration of M, or X0 is required to reach fmax; the method based on Eq. (4) or its analog, [ \aX,/af) vs. l/M,] is in this sense superior. Besides, Ii This
yellowish
color
is already
visnaily
noticeable
at. around
5 X 10“ M.
352
WATANABE,
FRANTZ,
AND
TROTTIER
[p-DIOXWIE]
FIG. 4. Effect [8-quinolinoll = tion of p-dioxane
of p-dioxane concentration on fluorescence of Mg*+-8-quinolinol. The concentra1.57 x lo-’ M and pH 8.9 - 9.2 (0.1 M Tris buffer), in $% (v/v) is shown at the right-hand side of each curve.
fO-3 FIG. 5. tration of 8-quinolinol linol and (ax&f) Tris buffer,
l
[X,
OR
Me]
Comparison of fluorescence increment upon addition of increasing concenMg*+ to a fixed concentration of 8-quinolinol with that upon addition of of 8-quinoto a fixed concentration of Mg*+. X0 f total concentration M. E total concentration of Mg*+. O,@: (i3M&f) versus l/Xa. A,A: versus l/MO. 0, A: 0.1 M triethanolamine buffer, pH 8.2. 0, A: 0.1 M pH 8.08.
FLUORESCENCE
OF
353
METAL-8-QUINOLINOL
@MO/V) or GXd~f) can be measured before the concenbration of formed complex QM exceeds 5 X 1O-5 iM, thus eliminating one of the two complications attended in the former method. This second method is illustrated in Fig. 5 with measurements at pH 8.1 + 8.2 using two different buffers (TEA and Tris), and with two symmetrical plots, i.e., (aM,/af) vs. l/X0 and (aX,/af) vs. l/M,. All four plots show a fairly good linear relation and have the same intercept on the vertical axis which gives a z 6.6 X 10’. This (Y value is smaller than that obtained previously (8 X 1O1) from the results shown in Fig. 2. This may not be due to the relatively high concentrations of S-quinolinol (lo--;’ to 10-l&1) used for measurements of (aM,,/af) since measurements of (aX,/aj, were made with 8 quinolinol in concentrations less than 5 X lo-” M and, as already pointed out, the plot of (aM,,/?f) vs. I/S,, coincided with the plot of (aX,/af) vs. l/M,. That the difference bet.ween the two CYvalues is, however, due to the difference in pH was indicated by the results shown in Table 1. The (Y value was obtained at, various pH’s from the intercept on the vertical axis in the plot of (?X,,/Zf) vs. (l/M,) and was observed to be dependent on pH. pH PH -(Y
DEPENDENCE
-.__
TABLE 1 OF (RELATIVE) MOLAR OF MG+-8-QUIXOLINOL
FLIX~RESCEXCE
!I 5
8.9
so
8.0
7.1
ti. 7
At 25” and in 0.1 M TEA
(~1
.-_---
7.2 --~.-6.2
buffer.
It should also be mentioned that the intercept on the horizontal axis in Fig. 5 gives the value for (-K’), 550 in Tris buffer (pH 8.1) and 650 in TEA (pH 8.2). Taking into account the difference in pH, the difference in K’ between Tris and TEA buffers is almost. negligible. The same situation will be seen later in Fig. 8b. b. At a sufficientIy low pH but not too low to avoid the participation of the ring ionization (pK s 5.05) and/or at a sufficiently low concentration of S-quinolinol, an approximation to Eqs. (4) or (5) is p GZ (l[aK’) (l/X,). Such a case is illustrated in Fig. 6, wherein p as a function of 1/X0 then passes through the origin and the reciprocal slope is &‘; CXK’ for Mg?+ GG3.2 X 10” liter mole-* at pH 7.15 (empty circles) and CYK’ for Ca?+ E 2.5 X 10” liter mole-l at pH 8.87 (solid circles). c. At high pH, e.g., 9.2, there arises a discrepancy between (ax,,, and (aM,), as shown in Fig. 7, in the presence of 80% (v/v) ethanol. While the magnesium concentration is lower than approximately 5 x lo+ M, a
354
WATANABE,
FRANTZ,
AND
TROTTIER
/
b -,'
0
I
0
4
I
I
I
6 I2 104* [x,-j -’
I
16
Fro. 6. Fluorescence increment per Mg” concentration and that per Ca*+ concentration at various concentrations of &quinolinol. X, E total ooncentration of 8-quinolinol. 0.1 A4 triethanolamine buffer. a: M,, E total concentration of Mg’+, pH 7.15. b: M. E total concentration of Ca’+, pH 8.87.
plot of (ax,/&) vs. (l/M,) shown in Fig. 7s provides the same values for K’ and (Yas obtained from a plot of (aM,/aj) vs. l/X, shown in Fig. 7b (solid circles) ; K’ E 1.08 x lo4 and Q E 8 X 105. However, when the magnesium concentration higher than 5 X 1O-sM was used, a plot of (ax&f) vs. (l/M,) gave a lower value for (Y and a higher value for K’: (YE 3.3 X IO5 and K’ r 3 X 104. This is possibly due to the existence of a species MQ2 or M,Q not taken into account in the foregoing analysis. It can be also seen in Fig. 7b that, when the concentration of B-quinolinol becomes so high that the absorption effect of yellow colored media is expected (see Fig. 3), a plot of (aM,/af) vs. (l/X,,) shows deviation from linearity. d. Taking account of (I) the light absorption effect by using B-quinolinol in the concentration less than 1O-3M and (B) the effect of high pH’s by using pH’s lower than 9, and (3) the pH effect on a by using the IInd type of experimentation rather than the Ist one, the K’ values for Mg*+ were estimated at various pH’s from the intercept on the horizontal axis in the plot of (aM,/af) vs. (l/X,,) and the results are summarized
FLUORESCENCE
OF
METAL-8-QUINOLINOL
355
FIG. 7. Effect of high concentrations of Mg” and 8-quinolinol at high pH and in (v/v) ethanol. MO E total concentration of Ma*+, X0 E total concentration of 8-quinolinol. pH 9.5 (0.1 M Tris buffer). The part of Fig. 7a framed by a dashed line is enlarged in 7b. 0: (ax&j) versus l/M,,, 0: @MO/a)) versus l/X,. 80%
in Fig. 8a. As described in the foregoing analysis, the intercept on the pH axis gives the value for (-log KK,,) : log KKH z -5.3. To obtain the KK, value, there is another way of experimentation based on Eq. (6). By use of a fixed and high concentration of 8-quinolinol (5 X lo-’ Af) and a low pH region (
It is solution complex ponents
assumed in the first place that the fluorescence observed in a containing 8-quinolinol and metal ions is completely due to a formed from one mole to one mole binding of these two com(i.e., the 1: 1 complex).
356
WATANABE,
FRANTZ,
AND
TROTTIER
Experimental observations are made of the fluorescence intensity as a function of the metal ion concentration as well as that of the 8-quinolinol concentration. The fluorescence is also st,udied as a function of pH. It is then shown that data obtained can be treated reasonably by adopting a well established reaction scheme for the binding of metal ions with 8-quinolinol:
For example, both a plot of (aM,/af) versus (l/X,) and that of (ax&f) versus (l/M,) are in a linear relation as long as the concentrations X0 and M,, are relatively low and both of them give the same value for (Y or K as expected from the assumption and the adopted scheme. The relation of (aM,,/af) or l/K to [H+] is shown (Fig. 8) to be also linear as expected from the adopted scheme and it gives the value for KK,(1fF3). The value of K, has been well established to be 1O-0,0at 20°C and zero ionic strength and 10-0.608at 25” and ionic strength 0.09 by Niislinen et al. [ (l), see also (7) 1. It is therefore a reasonable approximation to take lO-O.$ as the value for K,, under the experimental conditions used in this paper, i.e., 25” and ionic strength about 0.1. The K value for Mg2+-8-quinolinol is then calculated t.o be 10-.i.3/10-0.~ = 104,* With Ca*+, the values for a and K’ at pH 8.95 were estimated to be 211 x lo4 and lCP, respectively, from curve c in Fig. 3. If K, = 10-O.;, the K for Ca2+-8-quinolinol can be calculated to be K = 102.4(1 + 10°.7-8.05) = 103.‘. Furthermore, from slope b in Fig. 6, the value for LYK! was found to be 2.5 X 10fi at pH 8.87; if the (Yvalue obtained at pH 8.95 is adopted, K’ is found to be (2.5 X 10G)/(2.1 X lo*) = lO*.l at pH 8.87 and accordingly K s lo”.“. With Znz+, we have only approximate values because of the precipitation and low pH. From curve d in Fig. 3, (Y and K’ were approximated to be 2.1 X lo” and 105.6 at pH 7.4. From the latter, K for Znz+-8-quinolinol is estimated to be loo,‘-‘.” X 105.6 E 107.0. Furthermore, K’ for Zn’+ at pH 4.8 was approximately 103.’ in acetate buffer although (Yat this pH was found to be very small
FLUORESCENCE
OF
-LO8
[H’]
; pH 9
7
6
5
0
357
METAL-8-QUINOLINOL
9
IO [H+J
I6
I(109
FIG. 8. pH dependence of apparent binding constant (K’) for Mg’+ binding of 8-quinolinol and of the fluorescence increment at a fixed concentration of 8-quinolinol. A: upper left, --log K’ versus pH (0.1 M triethanolamine buffer, 0). B: lower right, (&Ff&lf) versus [H+1 (0.1 M Tris buffer, 0; 0.1 M triethanolamine buffer, A).
(0.33 x IO’). At this low pH, an ionization other than the phenol OH group has to be considered:
The dissociation constant of this ionization (KH~) was reported to be 1O5.01(1) at zero ionic strength and 20”. The observed K’ should therefore be:
K’ = K/(1 + [H+l/&
+ [H+12/PG&~l)
rather than : K’
ApproximaCng
=
WKH)/(KH
+
[H+l)
from the reported values (1) that
K, g 1O-y.’ and
358
WATANABE,
FRANTZ,
&I~ z lo+ under our experimental O.l), K is calculated to be: K -N
103.1
x
AND
TROTTIER
conditions 2 x
104.9 -N
(25’, ionic strength about 103.3
In Table 2, constants for metal binding of S-quinolinol thus obtained by us are listed together with those established by Niisanen and Pettinen (3). If one considers the difference in experimental conditions noted in the table, agreement of these two sets of constants is very good. MOLAR
FLUORESCENCE BINDING
TABLE 2 AND CONSTANTS FOR WITH 8-QUINOLINOL
Mg*+ a PK,’
pK,
/i = 0, 20” ,i = 0.1, 25’
0 N&&hen
and Pettinen
car+
7.0 (pH 8.95) 4.74
2.1
(pH
3.27 3.0 -3.2
4.4
METAL
ION ZIG+
8.95)
-2.1 (pH 7.4) 8.56 7.9 -8.3
(3).
As to the application of this fluorescence phenomenon to the magnesium determination, it should be mentioned that it is preferable from the viewpoint of sensitivity as well as specificity to use a high pH since KH for the phenol OH group of S-quinolinol is as high as 9.9 (1). Although there is an indication that complexes other than the 1: 1 complex are formed at high pH, fluorescence at any fixed concentration of 8-quinolinol increases linearly with increasing Mg2+ concentration, and therefore the high pH does not disturb the Mg2+ determination. Furthermore, Zn2+ interferes with the Mgz+ determination more at lower than at higher pH’s; the Zn2+ binding constant is so high, as compared with that of Mg”+, that Zn”+ still exhibits fluorescence at lower pH’s where Mgz+ cannot exhibit fluorescence, while at higher pH’s Zn2+ is possibly removed by precipitation and the molar fluorescence of the Zn2+ complex seems to be less than one-third that of Mg2+ complex. That Ca*+ interference with the Mg2+ determination is very little is understandable from our result that not only the Ca2+-binding constant is much smaller than that of Mg2+ but also the molar fluorescence of the Caz+ complex is less than one-third of that of the Ca*+ complex. SUMMARY
1. The study was made mainly in ayueous solutions of 0.1 M triethanolamine and tris (hydroxy) methylaminomethane on the fluorescence that was reported by Schacter in 1959 to be exhibited by an ethanolic solution of S-quinolinol and metal ions (Mg”, Ca?+, and Zn?+). The
FLUORESCENCE
359
OF METAL-8-QUINOLINOL
fluorescence was studied as a function of metal ion concentration, 8quinolinol concentration, and also pH. 2. The molar fluorescence and the apparent binding constant of these metal complexes and 8-quinolinol were then estimated. The apparent constants thus estimated were found to be in good agreement with those established by Nkiinen and Pettinen (1952) using the light absorption method, the potentiometric method, and the method of solubility equilibrium. 3. The sensitivity and specificity of the method for Mg’+ determination that was based on this fluorescence phenomenon were discussed in terms of the molar fluorescence and the binding constant. ACKNOWLEDGMENT
The authors wish to thank Dr. Manuel F. Morales stant encouragement and valuable discussion.
in this Institute
for his con-
REFERENCES 1. 2. 3. 4. 5. 6.
7.
N;~s~NEN, R., LUMME, P., AND MUKTJLA, A-L., Acta Chem. Stand. 5, 1199 (1951). N%;~NEN, R., Acta Chem. Stand. 5, 1293 (1951); see also 6, 352 (1952). NKSHNEN, R., AND PETTINEN, U., Acta Chem. Stand. 6, 837 (1952). SCHACTER, D., J. Lab. C&n. Med. 54, 763 (1959). BURTON, K., Biochem. J. 71, 3% (1959). STOCK, D. I., AND DAVIES, C. W., Truns. Faraday Sot. 44, 856 (1948) of the Metal Chelate Compounds.” MARTELL, A. E., AND CALVIN, M., “Chemistry
Prentice-Hall,
New York,
1952.
BIOCHRMISTRY
Fluorescence
5, 345-359
(1963)
of Magnesium-,
Calcium-,
quinolinol SHIZUO From
the
WATANABE,2 DONNA
Cardiovascular Medical
Zinc-8-
Complexes1 WILLIAM TROTTIER
Research Center, San Received
and
Institute, Francisco, July
FRANTZ,3 University California
of
AND
California
16, 1962
INTRODUCTION
About ten years ago, R. N&&en (l-3) made a series of extensive studies on the binding (or stability) constants of metal chelates of 8quinolinol, using the light absorption method, the potentiometric method, and also the method of solubility equilibrium. For example, the pk’ values at zero ionic strength and 20°C were reported to be 4.74, 3.27, and 8.56 for Mg”+, Ca?+, and Zn”+ binding, respectively (3). In 1959, D. Schacter (4) reported that an ethanol solution of magnesium-8-quinolinol exhibited fluorescence with a peak intensity at 530 ml*. He showed that this fluorescence was dependent on pH and specific for Mg’+. Among eleven other divalent cations he tested, Zn2+ was the only one that exhibited a significant intensity of fluorescence, i.e., approximately onehalf that observed with Mg?+. Ca2+ and Sr?+ exhibited negligible fluorescence and the other eight cations did not exhibit fluorescence. By utilizing these fluorescence characteristics, Schacter introduced a sensitive and specific method for magnesium determination in serum and urine. In the present paper, the fluorescence of Mg?+, Ca”+, and Zn’+ chelates of S-quinolinol is studied further in aqueous solutions (buffered with 0.1 M triethanolamine or 0.1 M tris(hydroxy)methylaminomethane) as well as in p-dioxane, which is found to be better than ethanol in increasing the sensitivity of the method; the results are analyzed in terms of the (relative) molar fluorescence and the binding constant. ‘This work was supported by grants from the American Heart Association and U. S. Public Health Service (H6085). ’ Established Investigator of the American Heart Association, Inc. 3The early part of this work was done while W. Frantz was on the Summer Research Fellowship of the American Physiological Society in 1960 at the Dartmouth Medical School, Hanover, New Hampshire. Present address: Department of Physiology, University of Michigan, Ann Arbor, Michigan. 4 Dartmouth Medical School, Hanover, New Hampshire. 345
346
WATANABE,
FRANTZ,
AND
TROTTIER
As for the sensitivity of the fluorescence method for Mg2+ determination, the molar fluorescence of Mg2+ chelate is shown to be 8 x loj (practical units-see definition in the section Materials and Methods) in p-dioxane. Even in aqueous solutions, it is found to be about 7 x IO4 and, accordingly, the sensitivity of this fluorescence method is still far superior to that of the light absorption method [the molar absorbancy = 2 x 103; cf. refs (2), (3), (5)], although the practical fluorescence unit could be compared to an absorbancy unit only in the practical purpose, depending on the commercially available instrument. As to the specificity of this fluorescence method for Mg*+, the molar fluorescence as well as the binding constant are found to make contributions to it. Ca*+-8-quinolinol as well as Zn2+-S-quinolinol is found to exhibit the fluorescence at 530 my upon activation at 360 rnp, but the molar fluorescence for both Ca2+ and Zn2+ chelates is found to be less than one-third of that for Mg2+ chelate. The pK values for metal binding with B-quinolinol obtained by this fluorescence method are 4.4, 3.1, and about 8.1 at an ionic strength of approximately 0.1 and 25°C for MgZ+, Ca2+, and Zn?+, respectively. These values are in good agreement with those reported by N&&en et al. (see above). The analysis that is used in this paper is therefore justified and the fluorescence method for Mg2+ determination gains theoretical basis as firm as the light absorption method. MATERIALS
AND
METHODS
An Aminco-Bowman spectrophotofluorometer equipped with a xenon photomultiplier tube was used to measure the fluorescence. Two slits with 0.5-cm openings were used, one before the activation light reached the 1 X 1 cm sample cell and the other before the emitted light reached the second monochrometer. The fluorescence intensity was expressed by the reading on the per cent transmission scale of the photometer (practical units). Using a mercury lamp as a calibration standard for the light wavelength, the fluorescence peak and the activation peak were observed to be at 530 and 360 rnp., respectively. With Zn?+ chelate, a little deviation in wavelength of the peaks was observed (515 and 375 mp) but all measurements were made of the fluorescence at 530 rnp with activation light of 360 mp. All chemicals used were commercially available and they were used without any further purification. S-Quinolinol and magnesium nitrate (6H,O) were Fisher certified reagents, and calcium chloride, zinc chloride, and magnesium chloride were Baker and Adamson standard purity reagents. Triethanolamine (TEA) 5 and tris (hydroxy) methyllamp and a IP28
s Abbreviations methylaminomethane.
used
are
TEA
for
triethanolamine
and
Tris
for
tris(hydroxy)-
FLUORESCENCE
OF
347
METAL-8-QUINOLINOL
aminomethane (Tris) were Fisher certified reagents; they were used at concentrations of 0.1 M as buffers. Sample solutions were kept in a constant-temperature bath of 25”, except for a few minutes for each measurement of the fluorescence intensity, when an aliquot volume (usually 3 ml) of sample solution was taken to the sample cell compartment which had no temperature control. TREATMENT
OF DATA
In the following discussion Mg”+ or Ca’+ will be denoted by M and undissociated 8-quinolinol by QH. The following equilibria are considered :
QHeQ+H+, M +&S&M,
&i
K
In particular, it is considered that conditions are always such that the equilibrium, M + 2 OH- = M(OH),, can be ignored (see 6). By combining Mass Law expressions, one obtains:
X,‘(Mo - X) (Xo - X) = &&/(KH
+ [H+l)
(1)
Here, the total concentrations of metal ions and 8-quinolinol are denoted by M, and X,,, respectively; the concentration of the complex QM by Xi; K, and K are a dissociation constant and an association constant, respectively, for the equilibria considered above. At constant pH the expression on the right-hand side of Eq. (1 J plays the role of an equilibrium const,wnt which we shall call K’. Differentiating Eq. (1) with respect to M, and combining the result with Eq. (Ii we obtain, at constant pH and X,: aS,‘aM,
= (X, - X)‘/(X/K’
+ (X, - X)/K’
+ (X, - X)?}
(2,
As M, is made very large, S + Xl,, and as M, is macle very small, X+ 0, hence from Eq. (2) it follows that the X(M,) curve will be of a “saturation” type, i.e., at very large M, it will be flat (slope = 0)) and near the origin its slope will be: gmo (aX/aMo)
= X0/( l/K’
+ XO)
(3)
Actually we measure not X, but the fluorescence, f, arising from excitation of QM. If we assume that f = olX (where (Y is the ‘
Equation (4) shows that a plot 01’ the limiting slope, /3, :Ig:Gnst (l/S,, 1
348
WATANABE,
FRANTZ,
AND
TROTTIER
is linear, that the intercept on the /3 axis (l/X, = 0) is l/a, and that the slope of the line divided by this intercept is l/K’. Recalling the definition of K' we may also re-express Eq. (4) as:
P = U/4 + l/bKXo) + t ll(cJ%J 1W&)[H+l Equation (5) shows that at constant X,, p is linear with [H+]. the intercept on the [H+] axis (p = 0) of such a plot, then: (-1~) Accordingly
and :
= &(I
(5) If IIt is
+ KXo)
(6)
: if KXO << 1, then (- IH) = if KXO = 1, then ( -IH) = if KY0 >> 1, then (- IH) =
KB, KH/2, KKHXo
Of course, it is also obvious from the definition of K' that, K' = K, when [H+] = KH, K‘ = K/2, and when [H’l <<&I, >>KH, K’ = K&I PI.
when [H+]
RESULTS
la. Keeping a fixed concentration of Squinolinol (X, = 1.92 x lo-” M) and varying the total concentration of added Mg2+ (n/r,), the intensity of fluorescence (f) at 530 rnp upon activation at 360 rnp was measured at various pH’s (various curves of Fig. 1). The resulting data are closely fitted by theoretical curves (solid lines of Fig. 1) based on the foregoing analysis, and therefore they justify said analysis. In curves such as those in Fig. 1, as M, is indefinitely increased it should be that X * X0 and f + fILlax, then from our assumptions f max = ax,,. In Fig. 2 are shown plots of fmar vs. X, in two different solvents; in such plots the slope is (Y. The data are, in fact, linear plots provided X, < 5 x 1O-5 M. In an aqueous solution (0.1 M triethanolamine buffer, pH 9.45), (Y = 8 X 104; in an ethanolic solution, 80% v/v (Tris buffer, pH 9.5)) a = 33 X 10”. When X, exceeds ca. 5 X 10e5M, the plots bend away from linearity; this is probably due to self-absorption by the increasing concentrations of the complex QM.‘i b. Equation (1) is symmetrical as regards M,, and X,,. Therefore if one starts with a fixed M, and increases X,, or starts with a fixed X,, and increases M,, the fmax a ttained in either case should be the same provided the starting M. equalled the starting X,. However, as shown in Fig. 3 (curves a and b), when we started with a fixed M. (2 X 10e5 M) ‘The fluorescence intensity is linearly with the stance only when the concentration is very low.
roncentration
of fluorescing
WI)-
FLUORESCENCE
349
OF METAL-8-QUINOLINOL
-4 LOG FIG. 1. Fluorescence as a function of Mg” concentration at various pH’s. Wquinolinoll = 1.92 x 10e5M, 0.1 M triethanolamine buffer, and 25”. Solid curves are calculated curves based on the analysis described in the text. Dashed curves are those tracing experimental results.
0
FIG. 2. Maximum intensity of S-quinolinol at 25”. Upper buffer). Lower curve: aqueous
0
4
0
f2
16
of fluorescence observed at various concentrations curve: 80% (v/v) ethanol and pH 9.5 (0.1 IIf Tris solution and pH 9.45 (0.1 M triethanolamine buffer).
350
WATANABE,
FRANTZ,
AND
TROTTIER
and increased X,,’ a lesser fmax was attained and, when the concentration X0 exceeded about 10-3.0 M, the fluorescence intensity decreased with
6
-4
-2
0
LOG [MO OR x0-j FIG. 3. Comparison of fluorescence intensity observed upon addition of increasing concentration of Mg’+ to 2 x lo-‘M &quinolinol with those of Ca2+ and Zn’+ and also with that observed upon addition of increasing concentration of Squinolinol to 2 x IO-“M Mg*+. Curve a: Mg*+ (M,) was added to 2 x lo-’ M 8-quinolinol in 0.1 M triethanolamine buffer, pH 8.95. Curve b: S-quinolinol (X0) was added t,o 2 x lo-‘M Mg’+ in 0.1 M triethanolamine buffer, pH 8.95. Curve c: Ca*+ (MO) was added to 2 x 10“ M 8-quinolinol in 0.1 M triethanolamine buffer, pH 8.95, Curve d: Zn’+ (M,) was added to 2 x 10-‘&f 8-quinohnol in 0.1 M triethanolamine buffer, pH 7.4. Mg” (curve a), Ca*+ (curve c), and Zn’+ (curve d) G M. were added to 2 x 10-‘J14 S-quinolinol in 0.1 M triethanolamine buffer (pH 8.95: curves a and c and pH 7.4: curve d). S-quinolinol (-X0) was added to 2 X lo-’ M Mg” in 0.1 M triethanolamine buffer, pH 8.95.
increasing X,,; this is probably not due to self-absorption of the formed complex QM since the concentration of QM in the presence of 2 X 10e5M magnesium (MO) cannot exceed 5 X 1O-5M, the concentration at which fluorescence of the complex began to show the self-absorption effect (See Fig. 2). This is also not due to the inadequacy of the considered equi‘A solution of metal ion (Me, Cal+, or Zn’+) without S-quinolinol certainly does not exhibit fluorescence but d-quinolinol solution did fluoresce without addition of metal ion. This fluorescence became significant at the concentration of &quinolinol approximately above 10e4 M, and was reduced only partially by addition of EDTA Therefore, this fluorescence is not due to the metal contamination of 8-quinohnol reagent. In curve b of Fig. 3, the difference between the fluorescence intensity with and without addition of magnesium ion was taken as the fluorescence intensity.
FLUORESCENCE
351
OF METAL-8-QUINOLINOL
libria, as will be indicated later in another type of very detailed experiments, but is probably due to absorption of both the activation light and fluorescence light by yellowish color of the S-quinolinol medium which was visually noticeable at these higher concentrations such as 10-“,“M,X or above. c. Bearing these complications in mind, we may estimate K’. For example, for curve a of Fig. 3, X, = 2 X 10-j M and pH 8.95. In this case, the half-maximum fluorescence (fmaxln) is obtained when [Mg*+] = 10-3.67 M. Such a condition is equivalent to setting X = X,/2 in the lefthand side of Eq. (l), i.e., to having K’ = l/Q!!, - X0/2) ; thus from the values just cited, K’ for Mg?+ = 103.G7liter mole-l at pH 8.95. Likewise, K’s for Ca*+ and for Zn2+ may be obtained from curves c and d; K’ for Ca?+ = 102,47 liter mole-l at pH 8.95 and X’ for ZrP+ = 1/(1O-4.s - 1O-5.u) s 1o”,6 liter mole-l at pH 7.4. With Zn2+, however, precipitation took place even when this low pH was used. Although measurements with Zn*+ were done quickly before precipitation became visible, the fact that the plateau had not been reached with curve d reflects the precipitation. The K’ value for Zn”+ cited above was therefore estimated on the assumption that the maximum fluorescence with Zn”+ is the same as that with Ca’+ (2.1 x lO’&). In order to avoid the precipitation with Zn?+, the pH had to be lowered to less than 5. Using 0.1 ill acetic acid-tetramethylammonium hydroxide, the half-maximum fluorescence was observed when [Zn?+] = lo-“.’ M at pH 4.8, indicating K’ for Zn’+ = lO’.l liter mole-’ at this pH. The maximum fluorescence observed at this pH was, however, very small-O.65 X 10’ for 2 X lo+ 1c1 S-quinolinol and accordingly a (Zn”+ chelated = 3.3 x lo3 at pH 4.8. Furthermore, Mg*+, even in :I concentration as high as 0.1 M, did not. produce any fluorescence in the r>reeence of 2 x 10e5M %quinolinol at this pH. Zn”+ chelate was therefore not studied further. d. cr (calculated from the slope of flllax vs. bl,, curves1 and 6’ (calculated from “half-saturation” values as above) were measured in waterethanol and water-p-dioxane solutions, at various pH’s (Fig. 4). The results (see also Fig. 1) can be summarized by saying that K’ is markedly reduced by pH but very little affected by adding p-dioxane; on the other hand, (Y is little affected by pH but markedly increased by p-dioxnnr. The effect of ethanol is qualitatively similar to that of p-dioxane. Ilu,. The method of estimating K’ by locating (fmaxlz) is simple but it does not fit for estimation at lower pH’s where a very high concentration of M, or X0 is required to reach fmax; the method based on Eq. (4) or its analog, [ \aX,/af) vs. l/M,] is in this sense superior. Besides, Ii This
yellowish
color
is already
visnaily
noticeable
at. around
5 X 10“ M.
352
WATANABE,
FRANTZ,
AND
TROTTIER
[p-DIOXWIE]
FIG. 4. Effect [8-quinolinoll = tion of p-dioxane
of p-dioxane concentration on fluorescence of Mg*+-8-quinolinol. The concentra1.57 x lo-’ M and pH 8.9 - 9.2 (0.1 M Tris buffer), in $% (v/v) is shown at the right-hand side of each curve.
fO-3 FIG. 5. tration of 8-quinolinol linol and (ax&f) Tris buffer,
l
[X,
OR
Me]
Comparison of fluorescence increment upon addition of increasing concenMg*+ to a fixed concentration of 8-quinolinol with that upon addition of of 8-quinoto a fixed concentration of Mg*+. X0 f total concentration M. E total concentration of Mg*+. O,@: (i3M&f) versus l/Xa. A,A: versus l/MO. 0, A: 0.1 M triethanolamine buffer, pH 8.2. 0, A: 0.1 M pH 8.08.
FLUORESCENCE
OF
353
METAL-8-QUINOLINOL
@MO/V) or GXd~f) can be measured before the concenbration of formed complex QM exceeds 5 X 1O-5 iM, thus eliminating one of the two complications attended in the former method. This second method is illustrated in Fig. 5 with measurements at pH 8.1 + 8.2 using two different buffers (TEA and Tris), and with two symmetrical plots, i.e., (aM,/af) vs. l/X0 and (aX,/af) vs. l/M,. All four plots show a fairly good linear relation and have the same intercept on the vertical axis which gives a z 6.6 X 10’. This (Y value is smaller than that obtained previously (8 X 1O1) from the results shown in Fig. 2. This may not be due to the relatively high concentrations of S-quinolinol (lo--;’ to 10-l&1) used for measurements of (aM,,/af) since measurements of (aX,/aj, were made with 8 quinolinol in concentrations less than 5 X lo-” M and, as already pointed out, the plot of (aM,,/?f) vs. I/S,, coincided with the plot of (aX,/af) vs. l/M,. That the difference bet.ween the two CYvalues is, however, due to the difference in pH was indicated by the results shown in Table 1. The (Y value was obtained at, various pH’s from the intercept on the vertical axis in the plot of (?X,,/Zf) vs. (l/M,) and was observed to be dependent on pH. pH PH -(Y
DEPENDENCE
-.__
TABLE 1 OF (RELATIVE) MOLAR OF MG+-8-QUIXOLINOL
FLIX~RESCEXCE
!I 5
8.9
so
8.0
7.1
ti. 7
At 25” and in 0.1 M TEA
(~1
.-_---
7.2 --~.-6.2
buffer.
It should also be mentioned that the intercept on the horizontal axis in Fig. 5 gives the value for (-K’), 550 in Tris buffer (pH 8.1) and 650 in TEA (pH 8.2). Taking into account the difference in pH, the difference in K’ between Tris and TEA buffers is almost. negligible. The same situation will be seen later in Fig. 8b. b. At a sufficientIy low pH but not too low to avoid the participation of the ring ionization (pK s 5.05) and/or at a sufficiently low concentration of S-quinolinol, an approximation to Eqs. (4) or (5) is p GZ (l[aK’) (l/X,). Such a case is illustrated in Fig. 6, wherein p as a function of 1/X0 then passes through the origin and the reciprocal slope is &‘; CXK’ for Mg?+ GG3.2 X 10” liter mole-* at pH 7.15 (empty circles) and CYK’ for Ca?+ E 2.5 X 10” liter mole-l at pH 8.87 (solid circles). c. At high pH, e.g., 9.2, there arises a discrepancy between (ax,,, and (aM,), as shown in Fig. 7, in the presence of 80% (v/v) ethanol. While the magnesium concentration is lower than approximately 5 x lo+ M, a
354
WATANABE,
FRANTZ,
AND
TROTTIER
/
b -,'
0
I
0
4
I
I
I
6 I2 104* [x,-j -’
I
16
Fro. 6. Fluorescence increment per Mg” concentration and that per Ca*+ concentration at various concentrations of &quinolinol. X, E total ooncentration of 8-quinolinol. 0.1 A4 triethanolamine buffer. a: M,, E total concentration of Mg’+, pH 7.15. b: M. E total concentration of Ca’+, pH 8.87.
plot of (ax,/&) vs. (l/M,) shown in Fig. 7s provides the same values for K’ and (Yas obtained from a plot of (aM,/aj) vs. l/X, shown in Fig. 7b (solid circles) ; K’ E 1.08 x lo4 and Q E 8 X 105. However, when the magnesium concentration higher than 5 X 1O-sM was used, a plot of (ax&f) vs. (l/M,) gave a lower value for (Y and a higher value for K’: (YE 3.3 X IO5 and K’ r 3 X 104. This is possibly due to the existence of a species MQ2 or M,Q not taken into account in the foregoing analysis. It can be also seen in Fig. 7b that, when the concentration of B-quinolinol becomes so high that the absorption effect of yellow colored media is expected (see Fig. 3), a plot of (aM,/af) vs. (l/X,,) shows deviation from linearity. d. Taking account of (I) the light absorption effect by using B-quinolinol in the concentration less than 1O-3M and (B) the effect of high pH’s by using pH’s lower than 9, and (3) the pH effect on a by using the IInd type of experimentation rather than the Ist one, the K’ values for Mg*+ were estimated at various pH’s from the intercept on the horizontal axis in the plot of (aM,/af) vs. (l/X,,) and the results are summarized
FLUORESCENCE
OF
METAL-8-QUINOLINOL
355
FIG. 7. Effect of high concentrations of Mg” and 8-quinolinol at high pH and in (v/v) ethanol. MO E total concentration of Ma*+, X0 E total concentration of 8-quinolinol. pH 9.5 (0.1 M Tris buffer). The part of Fig. 7a framed by a dashed line is enlarged in 7b. 0: (ax&j) versus l/M,,, 0: @MO/a)) versus l/X,. 80%
in Fig. 8a. As described in the foregoing analysis, the intercept on the pH axis gives the value for (-log KK,,) : log KKH z -5.3. To obtain the KK, value, there is another way of experimentation based on Eq. (6). By use of a fixed and high concentration of 8-quinolinol (5 X lo-’ Af) and a low pH region (
It is solution complex ponents
assumed in the first place that the fluorescence observed in a containing 8-quinolinol and metal ions is completely due to a formed from one mole to one mole binding of these two com(i.e., the 1: 1 complex).
356
WATANABE,
FRANTZ,
AND
TROTTIER
Experimental observations are made of the fluorescence intensity as a function of the metal ion concentration as well as that of the 8-quinolinol concentration. The fluorescence is also st,udied as a function of pH. It is then shown that data obtained can be treated reasonably by adopting a well established reaction scheme for the binding of metal ions with 8-quinolinol:
For example, both a plot of (aM,/af) versus (l/X,) and that of (ax&f) versus (l/M,) are in a linear relation as long as the concentrations X0 and M,, are relatively low and both of them give the same value for (Y or K as expected from the assumption and the adopted scheme. The relation of (aM,,/af) or l/K to [H+] is shown (Fig. 8) to be also linear as expected from the adopted scheme and it gives the value for KK,(1fF3). The value of K, has been well established to be 1O-0,0at 20°C and zero ionic strength and 10-0.608at 25” and ionic strength 0.09 by Niislinen et al. [ (l), see also (7) 1. It is therefore a reasonable approximation to take lO-O.$ as the value for K,, under the experimental conditions used in this paper, i.e., 25” and ionic strength about 0.1. The K value for Mg2+-8-quinolinol is then calculated t.o be 10-.i.3/10-0.~ = 104,* With Ca*+, the values for a and K’ at pH 8.95 were estimated to be 211 x lo4 and lCP, respectively, from curve c in Fig. 3. If K, = 10-O.;, the K for Ca2+-8-quinolinol can be calculated to be K = 102.4(1 + 10°.7-8.05) = 103.‘. Furthermore, from slope b in Fig. 6, the value for LYK! was found to be 2.5 X 10fi at pH 8.87; if the (Yvalue obtained at pH 8.95 is adopted, K’ is found to be (2.5 X 10G)/(2.1 X lo*) = lO*.l at pH 8.87 and accordingly K s lo”.“. With Znz+, we have only approximate values because of the precipitation and low pH. From curve d in Fig. 3, (Y and K’ were approximated to be 2.1 X lo” and 105.6 at pH 7.4. From the latter, K for Znz+-8-quinolinol is estimated to be loo,‘-‘.” X 105.6 E 107.0. Furthermore, K’ for Zn’+ at pH 4.8 was approximately 103.’ in acetate buffer although (Yat this pH was found to be very small
FLUORESCENCE
OF
-LO8
[H’]
; pH 9
7
6
5
0
357
METAL-8-QUINOLINOL
9
IO [H+J
I6
I(109
FIG. 8. pH dependence of apparent binding constant (K’) for Mg’+ binding of 8-quinolinol and of the fluorescence increment at a fixed concentration of 8-quinolinol. A: upper left, --log K’ versus pH (0.1 M triethanolamine buffer, 0). B: lower right, (&Ff&lf) versus [H+1 (0.1 M Tris buffer, 0; 0.1 M triethanolamine buffer, A).
(0.33 x IO’). At this low pH, an ionization other than the phenol OH group has to be considered:
The dissociation constant of this ionization (KH~) was reported to be 1O5.01(1) at zero ionic strength and 20”. The observed K’ should therefore be:
K’ = K/(1 + [H+l/&
+ [H+12/PG&~l)
rather than : K’
ApproximaCng
=
WKH)/(KH
+
[H+l)
from the reported values (1) that
K, g 1O-y.’ and
358
WATANABE,
FRANTZ,
&I~ z lo+ under our experimental O.l), K is calculated to be: K -N
103.1
x
AND
TROTTIER
conditions 2 x
104.9 -N
(25’, ionic strength about 103.3
In Table 2, constants for metal binding of S-quinolinol thus obtained by us are listed together with those established by Niisanen and Pettinen (3). If one considers the difference in experimental conditions noted in the table, agreement of these two sets of constants is very good. MOLAR
FLUORESCENCE BINDING
TABLE 2 AND CONSTANTS FOR WITH 8-QUINOLINOL
Mg*+ a PK,’
pK,
/i = 0, 20” ,i = 0.1, 25’
0 N&&hen
and Pettinen
car+
7.0 (pH 8.95) 4.74
2.1
(pH
3.27 3.0 -3.2
4.4
METAL
ION ZIG+
8.95)
-2.1 (pH 7.4) 8.56 7.9 -8.3
(3).
As to the application of this fluorescence phenomenon to the magnesium determination, it should be mentioned that it is preferable from the viewpoint of sensitivity as well as specificity to use a high pH since KH for the phenol OH group of S-quinolinol is as high as 9.9 (1). Although there is an indication that complexes other than the 1: 1 complex are formed at high pH, fluorescence at any fixed concentration of 8-quinolinol increases linearly with increasing Mg2+ concentration, and therefore the high pH does not disturb the Mg2+ determination. Furthermore, Zn2+ interferes with the Mgz+ determination more at lower than at higher pH’s; the Zn2+ binding constant is so high, as compared with that of Mg”+, that Zn”+ still exhibits fluorescence at lower pH’s where Mgz+ cannot exhibit fluorescence, while at higher pH’s Zn2+ is possibly removed by precipitation and the molar fluorescence of the Zn2+ complex seems to be less than one-third that of Mg2+ complex. That Ca*+ interference with the Mg2+ determination is very little is understandable from our result that not only the Ca2+-binding constant is much smaller than that of Mg2+ but also the molar fluorescence of the Caz+ complex is less than one-third of that of the Ca*+ complex. SUMMARY
1. The study was made mainly in ayueous solutions of 0.1 M triethanolamine and tris (hydroxy) methylaminomethane on the fluorescence that was reported by Schacter in 1959 to be exhibited by an ethanolic solution of S-quinolinol and metal ions (Mg”, Ca?+, and Zn?+). The
FLUORESCENCE
359
OF METAL-8-QUINOLINOL
fluorescence was studied as a function of metal ion concentration, 8quinolinol concentration, and also pH. 2. The molar fluorescence and the apparent binding constant of these metal complexes and 8-quinolinol were then estimated. The apparent constants thus estimated were found to be in good agreement with those established by Nkiinen and Pettinen (1952) using the light absorption method, the potentiometric method, and the method of solubility equilibrium. 3. The sensitivity and specificity of the method for Mg’+ determination that was based on this fluorescence phenomenon were discussed in terms of the molar fluorescence and the binding constant. ACKNOWLEDGMENT
The authors wish to thank Dr. Manuel F. Morales stant encouragement and valuable discussion.
in this Institute
for his con-
REFERENCES 1. 2. 3. 4. 5. 6.
7.
N;~s~NEN, R., LUMME, P., AND MUKTJLA, A-L., Acta Chem. Stand. 5, 1199 (1951). N%;~NEN, R., Acta Chem. Stand. 5, 1293 (1951); see also 6, 352 (1952). NKSHNEN, R., AND PETTINEN, U., Acta Chem. Stand. 6, 837 (1952). SCHACTER, D., J. Lab. C&n. Med. 54, 763 (1959). BURTON, K., Biochem. J. 71, 3% (1959). STOCK, D. I., AND DAVIES, C. W., Truns. Faraday Sot. 44, 856 (1948) of the Metal Chelate Compounds.” MARTELL, A. E., AND CALVIN, M., “Chemistry
Prentice-Hall,
New York,
1952.