Spectrophotometric and Spectrofluorometric Study of Flavonol-Aluminum Chelates in Absolute Ethyl Alcohol Fred L. Urbach‘ and Andrew Timnick Department of Chemistry, Michigan State University, East Lansing, Mich. 48823 In absolute ethyl alcohol, flavonol (3-hydroxyflavone) reacts with aluminum ions to form a series of fluorescent polynuclear chelates. Spectrophotometric evidence is presented for the existence of chelate species containing two aluminum ions per flavonol and one aluminum ion per flavonol. In addition, spectrofluorometric evidence indicates a 6:l aluminum to flavonol species. Potentiometric titrations of chelate solutions with ethanolic sodium hydroxide indicate that flavonol displaces two ethoxide ions from the coordination sphere of the aluminum ions in the formation of the 2:l aluminum to flavonol chelate. Further addition of flavonol proceeds by the displacement of one ethoxide ion and one ethanol molecule.
THENATURE of aluminum hydroxide species in aqueous solution and in the solid state has been investigated extensively. The polynuclear structure of y -A1(OH)3 has been determined by x-ray techniques ( I ) . In this crystalline form of aluminum hydroxide, six aluminum atoms, bridged by hydroxide ions, form a 12-membered cyclic unit. Crystalline basic salts of aluminum have been obtained by the addition of sulfate (2) or oxalate (3) anions to aluminum solutions containing up t o a 2.5-mole ratio of hydroxide ions t o aluminum. These salts have been shown t o contain a complex ion with the empirical formulation A12(OH)5f. Aluminum ions have been shown to exist as polynuclear species in aqueous solution. From studies of the titration of aluminum ion solutions with aqueous sodium hydroxide, Brosset, Biedermann, and Sillen (4) suggested that the predominant aluminum ion species before the equivalence point is A16(OH)ls3f. The nature of aluminum ion species in alcohol solutions, however, has not received much attention. That aluminum salts undergo extensive solvolysis in ethanol is apparent from the high acidity of the resulting solutions (5). The high charge to size ratio for aluminum ions leads t o extensive olation reactions (6). The potentiometric data from the titration of ethanol solutions of aluminum salts with sodium hydroxide yield a break corresponding to 2.5 hydroxide ions per aluminum (5). From this result, Ohnesorge has postulated the existence of a polynuclear aluminum ion species with the empirical formulation [Al2(0Et)# (5). The chelation reactions between aluminum ions and polyhydroxyflavones have served as the basis for photometric and fluorometric determinations of aluminum (7, 8). We (1) H. D. Megaw, Z . Krist., 87, 185 (1934). (2) G. Denk and L. Bauer, 2. Anorg. Allgem. Chern., 267, 89 (1952). ( 3 ) H. W. Kohlschutter and P. Hantelmann, ibid., 248, 319 (1941). (4) C. Brosset, G. Biedermann, and L. G. Sillen, Acta Chem. Scand., 8, 1917 (1954). (5) W. E. Ohnesorge and A. Capotosto, J. Inorg. Nucl. Cliem., 24,
829 (1962). (6) C. R. Rollinson in “Chemistry of the Coordination Compounds,” J. C . Bailar, Jr., Ed., Reinhold, New York, 1956, Chap. 13. (7) Z . G. Szabo and M. T . Beck, Acta Chim. Acad. Sei. Hunp., 4, 211 (1954). (8) C. E. White and C. S. Lowe, IND.ENG.CHEM.,ANAL.ED., 12, 229 (1940).
have investigated the chelation reaction between aluminum ions and flavonol (3-hydroxyflavone), and have found evidence concerning the nature of aluminum ion species in ethanol solutions. EXPERIMENTAL
Reagents. Reagent grade anhydrous aluminum chloride and sodium hydroxide and chemically pure absolute ethanol were used without further purification. Flavonol was synthesized by the method of Oyamada (9). The product was crystallized several times from aaueous alcohol and analvzed. Anal. Calcd for ClSH1003: C , 73.61 %; H , 4.23%. F o w d : C , 75.79%; H , 4.13%. Preparation of Stock Solutions. A 1.00 X lOW3Msolution of flavonol was prepared by dissolving 0.2382 gram of flavonol in 1 liter of absolute ethanol. Anhydrous aluminum chloride (0.3 gram) was dissolved in 250 ml of absolute ethanol t o give an approximately 0.01M solution. This solution was standardized according to a complexometric titration described by Schwarzenbach (IO). For successful titrations the aliquots of the stock solution had to be evaporated t o dryness and the residue dissolved in hydrochloric acid before proceeding with the standardization. A 1.00 x lOP3Msolution of AIC13 in ethanol was prepared by a n appropriate dilution of the standardized solution. Ethanolic sodium hydroxide solutions were standardized against aqueous solutions of potassium acid phthalate using phenolphthalein as the indicator. Instrumentation. All spectrophotometric data were obtained with a Cary Model 14 Recording Spectrophotometer. All spectrophotometric measurements were made with cells of I-cm path length. The fluorescence data were obtained with a spectrofluorometer constructed in our laboratory ( I I ) . The essential components consisted of a Hanovia Mercury arc source, a Bausch and Lomb excitation monochromator, and a Beckman Model DU spectrophotometer equipped with a photomultiplier tube as the emission monochromator and detector. The fluorescence spectra are reported without corrections for the variations in the emission characteristics of the source or the variations in detector sensitivity toward different wavelengths. Spectrophotometric and Spectrofluorometric Titrations. To simulate spectrometric titrations, the required volumes of the reagent solutions were added t o separate volumetric flasks and the solutions were diluted t o the mark with solvent. The measurements on the solutions in each flask represented a point on the spectrometric titration curves. Working solutions were sufficiently dilute to minimize errors caused by excessive absorption of the exciting radiation in the fluorescent measurements. The order of mixing of the reagents was critical so the flavonol solution was added to the Present address, Chemistry Department, Case Western Reserve University, Cleveland, Ohio 44106 (9) T. Oyamada, Bull. Chern. Soc. Japan, 10, 182 (1935). (10) G. Schwarzenbach, “Complexometric Titrations,” Interscience, New York, 1957, p 88. (11) L. L. Fleck, Ph.D. Thesis, Michigan State University, East Lansing, Mich. (1961). VOL 40, NO. 8, JULY 1968
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360
I
440
520
600
680
WAVELENGTH, mu
WAVELEN GTH, mu
Figure 1. Absorption spectra of flavonol and flavonolaluminum chelates
Figure 2. Fluorescence spectra of flavonol and flavonolaluminum chelates
A . 4.0 X 10-SMflav~p~l in absolute ethanol B. 4.0 X lO-5M flavonol with five-fold excess of aluminum(II1) in absolute ethanol
A. 4.0 X 10-6MfIavonolin absolute ethanol Excitation wavelength - 365 mp Emission monochromator slit = 1.8 mm B. 4.0 x lO-5M flavonol, 8.0 X lO-6M aluminum(II1) in absolute ethanol Excitation wavelength - 365 mp Emission monochromator slit = 1.0 m m
aluminum chloride aliquot, and this was followed by the addition of sodium hydroxide solution if required, If the hydroxide solution was added to the aluminum solution prior t o the addition of flavonol, the chelation reaction was severely inhibited. These solutions did not attain the same chelate absorbance values as comparable solutions mixed in the correct order, even after standing for 10 days. Apparent pH Measurements. A Beckman Zeromatic p H Meter with a combination glass-saturated calomel electrode was used to measure the apparent p H of the ethanol solutions. The meter was standardized with aqueous pH 7 buffer solution and the electrode was then allowed to equilibrate in absolute ethanol for 30 minutes before readings were taken.
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RESULTS AND DISCUSSION
Flavonal is capable of functioning as a bidentate chelate with a variety of metal ions by the use of the a-hydroxyketone functional groups. The changes in the absorption spectrum of flavonol which occur upon the addition of aluminum ions
(Figure 1) have been attributed to chelate formation (12). The bathochromic shift of the long wavelength absorption band by approximately 60 mp caused by the addition of aluminum ions has been reported to be characteristic of flavonols -i.e., flavones containing a 3-hydroxy group (12). Further evidence for chelation is given by the marked changes in the fluorescence spectrum of flavonol when aluminum ions are added (Figure 2). Flavonol solutions in ethanol exhibit two emission bands, a weak band at 410 mp and the major band at 535 mp. The emission spectra of the flavonol-aluminum chelates, regardless of stoichiometry, consist of a single band with maximum emission at 454 mp. Determination of Chelate Stoichiometries. Spectrophotometric titrations of aluminum chloride solutions with flavonol were employed to determine the stoichiometries of the chelates. The flavonol absorbance at 344 mp and the chelate absorbance a t 405 mp were followed in these titrations. In the absence (12) L. Jurd and T. Geissman, J. Oug. Chew., 21, 1395 (1956).
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ANALYTICAL CHEMISTRY
0
I 2 MOLE RATIO, F L A V O N O L
3
TO AI(III)
Figure 3. Spectrophotometric titration curves for aluminum(111)titrated with flavonol A . 0 2.0 X 10-SM in Aluminum(III), no added hydroxide; absor' bance measured at 344 mp B. 0 2.0 X 10-jM in Aluminum(III), no added hydroxide; absorbance measured at 405 mp C . A 2.0 X 10-jM in Aluminum(III), hydroxide-to-aluminumion ratio = 2.5 : l ; absorbancemeasured at 344 m p D. A 2.0 X 10-jM in Aluminum(III), hydroxide-to-aluminumion ratio = 2.5 : l ; absorbancemeasured at 405 mp
of added hydroxide, the spectrophotometric titration curves (Curves A , B, Figure 3) indicated that a 2 : l aluminum t o flavonol species was formed. On standing, further chelation occurred slowly in these solutions and after two weeks the indicated stoichiometry was 1 : I . The existence of a 2 : l aluminum to flavonol species in solutions to which no hydroxide was added was confirmed by a spectrophotometric titration of flavonol with aluminum ions and by the method of continuous variations. Because it is most likely that flavonol acts as a bidentate chelating agent toward one metal ion and has little tendency t o function as a bridging ligand, the unusual stoichiometry of these species reflects the binuclear or polynuclear character of the aluminum ion species. Evidence is presented below that the aluminum ion aggregates contain more than two aluminum ions.
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1 2 3 MOLE RATIO, FLAVONOL TO A I ( I I I )
4
Figure 4. Typical fluorometric titration plot for aluminum(II1) titrated with flavonol 2.0 X 10-5Min Aluminum(III), no hydroxide added Excitation wavelength - 365 mp Emission monochromator slit = 1.6 mm
A . 0530mp B. O450mp
T o promote chelate formation, ethanolic sodium hydroxide was added to the flavonol-aluminum ion solutions to neutralize the protons released by the solvolysis of the metal ions and by the chelation reaction. When the spectrophotometric titration of aluminum ions with flavonol was carried out with added sodium hydroxide to give an OH/A1 ratio of 2.5 :1, a 1 :1 aluminum-flavonol species is formed (Curves C, D, Figure 3). In these solutions there was no change in the indicated stoichiometry with time. In Curve D the continued increase in absorbance values at 405 mp beyond the end point may be attributed to the conversion of a portion of the flavonol to the anion form in the presence of hydroxide. This flavonol anion has an absorbance maximum at 412 mp and thus contributes to the absorbance values at 405 mp. When the amount of sodium hydroxide added was increased to give an OH/Al ratio of 3 :1 or greater, the chelation of aluminum ions by flavonol was severely inhibited, and is attributed to an unfavorable competition with the excess hydroxide ions for the coordination sites of the metal ion. No breaks were indicated in the curves for the spectrophotometric titrations carried out at these hydroxide-to-aluminum ratios. The fluorescence of the solutions employed for the spectrophotometric titrations was measured at 450 mp and 530 mp, both excited by 365-mp radiation. These data are presented as spectrofluorometric fitrations in Figure 4. The emission at 450 mp may be attributed primarily to the chelate species with a small contribution from the uncomplexed flavonol emission. At 530 mp the emission is essentially that of the uncomplexed flavonol with some contribution from the chelate species. The titration curve following the flavonol emission at 530 mp (Curve A , Figure 4) yielded a break corresponding to the 2 :1 aluminum-to-flavonol species detected by the spectrophotometric titration. In addition, the fluorescence titration curve exhibited a small peak prior to the 2 : l break due to the contribution of an intense chelate emission. This chelate species is identified below. In this titration there was no added hydroxide. The curves obtained by following the emission at 450 mp in these titrations were anomalous (Curve B, Figure 4). An intense emission peak was exhibited when the flavonol-toaluminum ratio was between 0 and 0.5. This result in-
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108 6 4 MOLE RATIO, AI(III1 TO FLAVONOL
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Figure 5. Spectrofluorometric titration curves for aluminum(II1) titrated with flavonol to indicate the existence of a 6:l aluminum-to-flavonol species 8.0 X 10d5Min Aluminum(II1) ions Emission monochromator slit = 0.38 mm 0 Excitation wavelength - 365 mp 0 Excitation wavelength - 405 mp
dicated the existence of a highly fluorescent species with a high aluminum-to-flavonol ratio. The spectrofluorometric titration curves obtained when the OH/Al ratio was 2.5 :1 did not yield interpretable breaks but did indicate a highly fluorescent chelate species as above. When the hydroxide-to-aluminum ratio was 3 :1 or greater, any flavonol-aluminum chelates which formed no longer fluoresced. The only fluorescence observed in these solutions was the flavonol emission at 530 mp and no breaks were indicated in these titration curves. Spectrqfluorometric titrations of aluminum ions with flavonol (Figure 5 ) and flavonol with aluminum ions (Figure 6) were carried out in the region of the intense chelate fluo-
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2 4 6 8 MOLE RATIO, AI(II1) TO FLAVONOL
10
Figure 6. Spectrofluorometric titration curve for flavonol titrated with aluminum(II1) to indicate the existence of a 6:l aluminum-to-flavonolspecies 2.0 X 10 -5M in Flavonol
Emission monochromator slit = 0.30 mm Excitation wavelength - 365 mp VOL. 40, NO. 8, JULY 1968
0
1271
rescence to determine the stoichiometry of the highly fluorescent species indicated in the above titrations. These titrations indicated the existence of a species containing six aluminum ions to one flavonol. In both of these titrations the flavonol solution was added to the aluminum chloride aliquot to maintain a t all times a n excess of aluminum ions t o prevent the formation of the 2 :1 aluminum-to-flavonol chelate which may preclude the formation of the 6 :1 species. There was no hydroxide added in either of these titrations. The chelate absorbance at 405 mp was measured in both of these titrations, and in both cases no indication of a 6 : 1 species was obtained from the spectrophotometric data. The fact that the spectrofluorometric data indicate the existence of a 6 :1 aluminum-to-flavonol species, whereas the spectrophotometric data d o not indicate this species, can probably be rationalized by the difference in the nature of absorbance and fluorescence. A variation in stoichiometry will only be indicated by a spectrophotometric titration when there is a large enough difference in the stabilities of successive complexes and if the absorption characteristics of the species vary. On the other hand, the emission of a chelate is much more sensitive t o the environment of the metal ion. If successive chelates have enough difference in the fluorescence characteristics, the spectrofluorometric titration curve will yield a break even though no break is indicated from the complementary absorbance data. Potentiometric Titrations of Chelate Solutions with Hydroxide Ions. Aluminum ions in absolute ethanol may be titrated potentiometrically with hydroxide ions. The results of such a titration (Curve A , Figure 7 ) are in agreement with the results of Ohnesorge (5) in which a well-defined end point occurs at an OH/Al ratio of 2.5 : I . I n the titrations of aluminum ion solutions and chelate solutions there were n o indications of precipitate formation. Ohnesorge postulated on the basis of these titration data that the simplest aluminum ion species in ethanol at the end point of the titration must be a binuclear species bridged by solvent molecules or solvent ions.
ItOb
'
'
I I 2' 3I I 4 I MOLE RATIO, OH' TO AI(III)
Figure 7. Potentiometric titration curves for aluminum(II1) and a flavonol-aluminum chelate titrated with hydroxide ions in absolute ethanol A . 0 2.0 X 10-4M in Aluminum(II1) B. 0 2.0 X 10-'M in Aluminum(III), 1.0 10-4Minflavonol
flavonol must release a proton when it chelates with a metal ion, in order for there to be a net loss of titratable protons in the solution, the flavonol must displace two ethoxide ions from the solvated aluminum ion species. If one ethanol molecule and one ethoxide ion were displaced by a n incoming flavonol, there would be no net change in the titration curve. The displacment of two ethanol molecules would result in more base being required to titrate the chelate solutions. The overall reaction of the aluminum species with flavonol in which the number of protons available is decreased may be summarized as : [Al,(OEt)j(EtOH),]+
+ 5Hf + FlOH
4
[Al,(OEt),(EtOH),(FIO)]*+
With the addition of flavonol to the aluminum solutions, a n additional break in the potentiometric titration curve appears before the OH/Al ratio reaches 2.5 :1. This new break is first apparent when the aluminum-to-flavonol ratio is 4 :1 . Increasing the amount of flavonol present to give a ligand-toaluminum ion ratio of 1 :2 shifts this first break to an OH/Al ratio of 2.0:l (Curve B, Figure 7). Further addition of flavonol does not shift this break below this value but does cause it t o be better defined. In all of the titrations of chelate solutions a second break occurs at an OH/Al ratio of approximately 2.7 : 1. Because less base is required to titrate flavonol-aluminum chelate solutions than is required t o titrate the aluminum ion solutions, it is concluded that the interaction of flavonol with aluminum ions in some way neutralizes protons. Because
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ANALYTICAL CHEMISTRY
x
+ 4H+ + 2EtOH
where FlOH represents 3-hydroxyflavone. The subsequent break in the titration curves for chelate solutions is viewed as the removal of a proton from the complex species t o yield a singly charged cation.
+
[A1~(OEt)~(EtOH)n(F10)]2+OH-
+
[A12(0Et)~(EtOH),-i(FlO)]++HzO Further addition of flavonol to the complex species t o form the 1 :1 aluminum-to-flavonol chelate must occur by the displacement of one ethoxide and one ethanol, as the position of the end point in the titration curve does not shift below a 2 :1 OH/AI ratio even in the presence of excess flavonol. RECEIVED for review January 29, 1968. Accepted April 8, 1968. The authors are grateful for the support provided to F. L. Urbach through National Science Foundation and Socony-Mobil Fellowships.
