fluorescence studies of the effects of t-butyl functionalities on the formation of ternary...

Fluorescence Studies of the Effects of t-Butyl Functionalities on the Formation of Ternary 13-Cyclodextrin Complexes with Pyrene

V I N C E N T C . A N I G B O G U a n d I S I A H M . W A R N E R *

Department of Chemistry, Clark Atlanta University, Atlanta, Georgia 30314 (V.C.A.); and Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803 (1.M.W.)

The effects of t-butyl (TB) compounds on the formation of ~ - c y -

c l o d e x t r i n (CD)/pyrene complexes have been examined by monitoring changes in the fluorescence I/III band ratio of 1.0 x 10 7 M

pyrene in 0.075 M of the t-butyl compounds and in various concentrations of ~-CD in a solvent mixture consisting of 60% v/v methanol/40% water solution. For the compounds tested, the strongest effects were observed in the presence of the following (in decreasing order): TB-carbamate, TB-carbazate, TB-(N-hydroxy) carbamate, TB-(N-butoxy carbonyl) glycine, and TB-formate. In terms of enhancement of the pyrene/13-CD binding constant, all exhibited effects stronger than those previously reported for t-butyl alcohol. In contrast, TB-amine, TB-acetic acid, and TB-acetoacetate exhibited very little effect. There appears to be a relationship between the chemical structure of the modifier molecules and the magnitude of their concomitant effects On the I/III ratio. In fact, the t-butyl modifiers which exhibited moderate to strong effects have a common caboxy, - C - O - , skeleton attached to the t-butyl moiety in their general structure: (CH3)3C-O-R. The modifier effect was also found to be independent of the pH of the solutions. A detailed explanation has been offered for the observed changes in the I/III ratio relative to t-butyl functionalities.

Index Headings: Steady-state fluorescence; [3-cyclodextrin; Pyrene; Modifier effects; Tertiary-butyl compounds.

I N T R O D U C T I O N

Cyclodextrins (CDs) are naturally occurring cyclic oli- gosaccharides that have the unique ability to form stable inclusion complexes with a variety of organic and inor- ganic molecules and ions / The inclusion phenomenon of the CDs has found applications in the pharmaceutical, food, and cosmetic industries. 2-4 Such industrial applications are expected to increase in the future once large- scale uses for CDs are approved by the Food and Drug Administration. Applications of CDs in analytical chemistry mostly relate to the development of analytical meth- odologies for separation, extraction, isolation, and improved detection of various analytes? -7 Successful ex- ploitation of the CDs in application designs and meth- odology developments requires good knowledge of the underlying inclusion mechanisms and the associated stoi- chiometries and formation constants, s Such knowledge can be gained only through detailed fundamental studies of the CD inclusion chemistry.

The degree to which a molecule is complexed by CDs depends on the size, the polarity, and the chemical nature of the substituents on the guest, as well as the pH and ionic strength of, and the presence and concentration of

Received 21 August 1995; accepted 18 March 1996. * A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d b e sent .

concomitants in, the medium. The concomitant effects of organic compounds as solution modifiers on the formation of 13-CD/guest complexes have been the subject of many studies. 9-2° For example, the concomitant effects of some alcohols including t-butanol on the [3-cyclodextrin inclusion of pyrene have been studied by using steady- state fluorescence measurements. 9-~3 With the use of the variation of the I/III fluorescence band ratio of pyrene, these studies have shown that pyrene forms markedly more stable complexes with [3-CD in water to which --< 1% v/v t-butyl (TB) alcohol or cyclopentanol has been added than it does in water alone. 15,~6 All the soluble lin- ear and cyclic alcohols studied exhibited such increases in the formation of the [3-CD/pyrene complex. In another study, Mufioz de la Pefia et al. ~7 have also shown that pyrene, which forms unstable complexes with [3-CD in > 4 0 % v/v methanol /<60% water mixtures (a typical reversed-phase HPLC mobile-phase composition), forms markedly stable complexes with [3-CD (Kr "~ 105) in these methanol/water solutions to which _<2% v/v cyclopentanol or t-butyl alcohol has been added. Formation of a 2:1:2 ternary complex of modif ier /pyrene/#-CD has been postulated to explain the observed enhancement? 5,16

Anigbogu et al. 18,19 conducted reversed-phase HPLC studies of the pyrene/[3-CD system under conditions similar to those for the fluorescence studies, and they ob- tained similar results vis-a-vis the improved inclusion of pyrene by t3-CD in the presence of some t-butyl compounds or cyclopentanol. However, anthracene, which forms a more stable [3-CD complex (Kf ~ 250) than pyrene in these methanol/water mixtures, shows no evi- dence of enhanced c o m p l e x a t i o n upon addi t ion of t-butanol as the co-modifier. ~8,~9 This observat ion implies that the concomitant effect of these co-modifiers on the [3-CD/guest inclusion chemistry is highly selective. Re- cently, Husain et al? ° reported an HPLC study on the concomitant effects of some t-butyl compounds on the formation of 13-CD/pyrene complexes. The effects of se- lect t-butyl compounds, with functional groups of varying polarity and heteroatomic composit ion, on the formation of [3-CD/pyrene complexes were examined by monitoring the changes in the retention times of pyrene in reversed-phase HPLC. The results show that the type and the polarity of the functional groups attached to the t-butyl moiety of the secondary modifier appear to significantly affect the equilibrium of pyrene between the C-1 8 stationary phase and the methanol/water/[3-CD mobile phase.

The reversed-phase HPLC technique involves long re-

Volume 50, Number 8, 1996 0003-7028/96/5008-099552.00/0 APPLIED SPECTROSCOPY 995 © 1996 Society for Applied Spectroscopy

TABLE I. Structures of t-butyl and other compuonds whose effects 1.44 on the formation of ~-CD/pyrene complex were examined.

Name Structure

1. t-Butyl alcohol 2. t-Butyl amine 3. t-Butyl formate 4. t-Butyl acetate 5. t-Butyl acetoacetate 6. N-(t-butoxycarbonyl)

glycine 7. t-Butyl acetatic acid 8. t-Butyl carbamate 9. t-Butyl carbazate

10. t-Butyl N-hydroxy carbamate

(CH3)3C-OH (CH3)3C-NH 2 (CH3)3C-O-CO-H (CH3)3C-O-CO-CH 3 (CH3)3C-O-CO-CH2-CO-CH 3

(CH3)3C-O-CO-NH-CH2-CO-OH (CH3)3C-CH2-CO-OH (CH3)3C-O-CO-NH 2 (CHs)3C-O-CO-NH-NH z

(CH3)sC-O-CO-NH-OH

tention times for pyrene, consumes large quantities of solvents, and generates large quantities of waste, which precluded an extensive study of t-butyl modifiers of various functionalities. This paper represents a more com- prehensive, systematic examination of the effects of t-butyl compounds on the formation of [3-CD/pyrene complexes using steady-state fluorescence measurements. The modifiers studied, including those in Ref. 20, are listed in Table I.

EXPERIMENTAL

Apparatus. Steady-state fluorescence measurements were performed with a Spex-fluorolog-2 Model F2T21I computer-controlled spectroflorometer (Spex Industries, Edison, NJ) equipped with a cell compartment thermos- rated at 25 ± 1 °C with a VWR Model 1160 constant- temperature circulator. With excitation and emission bandwidths set at 5 and 3 nm, respectively, solutions were excited at 335 nm, and emission spectra were ac- quired from 350 to 420 nm. Each spectrum was an av- erage of three scans, and no further smoothing was performed.

Reagents. Pyrene (99+%), all the t-butyl compounds (99+%), and cyclopentanol (99+%) were purchased from Aldrich and were used as received. The methanol (Baxter, McGraw Park, IL) and water (Fisher, Fair Lawn, NJ) used were both HPLC grade. The [3-cyclodextrin was a gift from American Maize Products (Hammond, IN) and was also used as received. Buffers were made with Fisher (Fair Lawn, NJ) reagent grades of the following compounds: KH2PO 4, Na2HPO 4, NaHCO3, Na2CO3, KC1, and HC1.

Methods for Sample Preparation. Procedure for the Preparation of Pyrene Solutions. A 250-mL stock solution of 1.0 × 10 -4 M pyrene was first prepared by dis- solving 5.07 mg of pyrene and diluting to 250 mL with methanol. A 250-mL working solution of 1.0 × 10 5 M pyrene was prepared from the stock solution.

Effect of t-Butyl Compounds. A 100-~L aliquot of 1.0 × 10 -s M pyrene was pipetted with an Eppendof pipette into a pre-cleaned 10-mL volumetric flask. Appropriate amounts of t-butyl compounds and D-CD that would give, upon dilution, 0-0.10 M and 0 - 6 mM concentrations were dissolved and diluted to the mark with 60% v/v methanol/40% water solution. The flask was sealed with parafilm, and the contents were sonicated for 30 rain at 25 °C.

0

m

1,33

1 . 2 2

1.11

\

1 . 0 0 • - . . .

0 . 8 9 q i i ~ " l

0 . 0 1.3 2,5 3.8 5,0 6 . 3

[B-CD], mM

FIG. 1. Changes in the I/III vibronic band ratio of pyrene vs. 13-CD concentration in the presence of 0.075 M modifiers: blank (methanol/water) (0); TB-alcohol (U); cyclopentanol (l); TB-amine (V); cyclopentylamine ( • ).

Effects of pH on the Modifier Effect. Procedures for the prepara t ion of buffer solut ions in me thano l /wa te r mixtures have been published? ~,22 One-liter buffer solutions of similar ionic strength were prepared in a mixture of 60% v/v methanol/40% v/v water by adding the following: (1) pH 2 :2 .0 mL of concentrated HC1 and 0.7456 g of KC1; (2) pH 5 : 1 . 0 mL acetic acid, 0.50 mL of 50% solution of NaOH, and 0.745 g of KC1; (3) pH 7 :0 .346 g KH2PO4, 0.358 g Na2HPO4, and 0.7465 g KC1; (4) pH 9 : 1 . 9 0 8 g Borax and 0.747 g KC1; (5) pH 11 :0 .210 g NaHCO3, 0.265 g Na2CO 3, and 0.745 g KC1. The final pH of the solution was checked with a pH meter.

RESULTS AND DISCUSSION

Effects of t-Butyl Compounds on the I/III Ratio. The fluorescence spectrum of pyrene monomer is com- posed of vibrational fine structures, whose relative band intensities are affected by its microenvironment. 23-26 It has been shown that the ratio of the first band to the third or the fifth (I/III or I/V) depends on the polarity of pyrene's microenvironment. For example, at the emission slit width of 3 nm used in this study, the I/III ratio of pyrene is ~ 1.5 in pure water, ~0.70 in an aqueous solution containing 2.5 × 10 3 M of S-CD, and ~0.6 in cyclohex- ane] 5,16 In water/methanol mixtures, the [/III ratio ranges from ~1.5 in 0% methanol to ~1.3 in 70% methanol. The band ratio technique of pyrene, among other probes, has become a sensitive technique for probing the polarity of microenvironments such as the cyclodextrin cavity. 17 It should be mentioned that the I/III ratio of pyrene in water reported here (~1.5) is slightly lower than the literature value of ~1.8. The difference stems from differ- ences in the slit width settings. Our measurements were made with a 3-nm slit width setting, while the literature value was determined with a 1-nm slit width. However, since we are interested in using relative changes in the I/III ratio only as a means of monitoring the degree of inclusion of pyrene by [~-CD in the presence of different t-butyl compounds as modifiers, the difference in I/III ratio values has no effect on the experimental outcome.

Figure 1 shows plots of the I/III ratio vs. the concen-

996 Volume 50, Number 8, 1996

1.4

1.3

1.2

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1,0

0,9 0.0

0

_=

|

(

~ - . . \ + ~ - - .

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[B-CD], mM

F]G. 2. Changes in the I/III vibronic band ratio of pyrene vs. [3-CD concentration in the presence of 0.075 M modifiers: TB-alcohol (11); TB-formate (IS]); TB-carbamate (O); TB-carbazate (+); TB-N-hydroxycarbamate (S); N-(t-butoxycarbonyl) glycine (V); and cyclopentanol (~).

tration of [3-CD for solutions containing 1.1 × 10 -v M pyrene and 0.075 M concentrations of TB-alcohol, TB- amine, cyclopentanol, and cyclopentylamine, respectively. The solutions were prepared in a solvent mixture comprised of 60% v/v methanol in HPLC-grade water. The change in the I/III ratio in methanol/water (blank) solution is included for reference. At zero [3-CD concentration, the value of the I/III ratio of pyrene ranges from --~ 1.30 to --~ 1.39 in these solutions, which agrees with our previous report.Iv However, as the concentration of [3-CD is changed f rom 0 to 6 mM, the attendant changes in the I/III ratio are insignificant in water/methanol solution and in TB-amine-modif ied or cyclopentyl amine-modified solutions. As expected, significant changes in the I /III ratio were observed both in the cyclopentanol- or t-butyl alcohol-modified solution, in keeping with previous obser- vations. 15-1~ Although not shown in this report, I / III changes in 0.075 M solution modified by TB-acet ic acid, TB-acetoacetate, and trienthanol amine, respectively, were similar to those in water/methanol and t-butyl amine systems. These results are not reported because of the unreliability of the data as a result of incomplete disso- lution of [3-CD in these solutions. The problems were persistent at [[3-CD] above 3 mM even after sonication for one hour at 40 °C. We have previously reported a correlation between [3-CD solubility in the presence of a given modifier and the magnitude of the formation constant for the [3-CD/pyrene/modifier ternary complex. ]8,19 This observat ion implies that the mechanism(s) of the concomitant effects may involve, among others, a solubility effect.

P rev ious s tudies show that t -bu tano l , o f all the branched soluble alcohols examined, exhibited the great- est effect on the fluorescence I/III band ratio of pyrene. L5,~6 The data plotted in Fig. 1 suggest that the functional group attached to the t-butyl moiety may also play an important role in the development of the concomitant effects. Figure 2 shows more plots of the I/III ratio vs. the [3-CD concentration in the presence of TB-formate, TB-carbamate, TB-carbazate, TB-N-hydroxycarbamate ,

FIe. 3. Depiction of molecular interactions that may be responsible for the formation of [3-CD/pyrene/TB-modifier ternary complexes with a 2:1:2 stoichiometric ratio: R = t-butyl moiety; R~ = polar group attached to the - C - O - skeleton, which is capable of hydrogen bonding.

and N-(t-butoxy carbonyl) glycine. The data for TB-alcohol and cyclopentanol are included for comparison. As indicated by the slopes of these curves, all data for these TB-compounds suggest effects that are stronger than that of t-butanol. In fact, close examination of the structures of the t-butyl compounds in Table I, which exhibit ]nod- crate to strong effects (Figs. 1 and 2), reveals a common carboxy, - C - O - , skeleton attached to the t-butyl group in the general structure: (CH3)3C-O-R.

In explaining the mechanism for the concomitant effects of cyclopentanol on the formation of [3-CD/pyrene complexes, Mufioz de la Pefia et al. 15,16 postulated two mos t p r o b a b l e conf igura t ions for the [3-CD/pyrene/ cyclopentanol complex with a 2:1:2 stoichiometric ratio. I f this argument is extended to t-butyl compounds, then positioning the modifier molecules at the open ends of the cyclodextrin molecules means that the t-butyl (the hydrophobic) end will be partially or completely encapsulated within the [3-CD. This configuration is depicted in Fig. 3. The attached - R group, if polar in nature and able to form hydrogen bonding, would interact favorably with the solution (methanol/water) medium. This ar- rangement would leave the - C - O - moiety on the modifier at or near the edge of the [3-CD, hence, allowing interaction with the secondary hydroxyl groups on the [3-CD. Obviously, the magnitude of the observed modifier effects would be determined by how well these forces counterbalance, which, in turn, depends on the modifier chemical structure. For example, TB-acetate has the - C - O - moiety attached to the t-butyl, (CH3)3-, group while TB-acetic acid does not. As a result, a significant difference was observed for the effects of 0.075 M of TB- acetic acid and 0.075 M TB-acetate as modifiers. This observation perhaps illustrates the importance of hydrogen-bonding interactions between the secondary hydroxyl groups at the edges of [3-CD and the functional groups attached to the t-butyl moiety. The stronger effect shown by TB-formate than TB-acetate demonstrates the importance of hydrogen bonding between the modifier molecule, [3-CD, and/or the bulk (methanol/water) solution. The extreme case of this situation is observed in the presence of TB-acetoacetate, where the - R group is less polar and much less likely to participate in hydrogen bonding with the bulk solution than the - R group in the TB-ac- crate; this behavior results in little or no effect. As for the modifier molecules that lack the - C - O - R moiety, as is the case with TB-acetic acid and TB-acetoacetate, no significant effects are observed in the form of changes in the I/III ratio. This result again suggests a lack of hydrogen-bonding interaction among the modifier molecules,

APPLIED SPECTROSCOPY 997

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FIG. 4. Influence of modifier concentration on the I/III ratio of pyrene in the presence of 3 mM [3-CD/TB-carbamate (O); TB-carbazate ( • ) ; N-(t-butoxycarbonyl) glycine (1); TB-N-hydroxycarbamate (+); cyclopentanol (V); and TB-butanol (O).

the solution medium, and/or the secondary hydroxyl groups of the [3-CD. If, however, the modifier molecule contains an - R group, such as in TB-amine and TB-acetoacetate, which is less likely to participate in hydrogen bonding with the bulk solution, the I/III ratio changes only slightly and the [~-CD solubility decreases dramatically. This result suggests that pyrene is minimally encapsulated by [3-CD in the presence of these species even at higher [~-CD concentrations. This result also strongly points to the formation of a more stable modifier/[3-CD complex than the pyrene/[3-CD complex, especially since the modifier is present at a concentration level (0.075 M) about five orders of magnitude greater than that of pyrene ( 1 . 0 X l0 7 M ) . A comparison of the plots for cyclopentanol and cyclopentylamine (Fig. 1) further supports this argument.

It appears that three competing forces are important in the concomitant effects of modifiers in the formation of [3-CD/pyrene/modifier ternary complexes, namely: (1) the hydrophobic interaction of the t-butyl moiety with the high electron density of the glycosidic oxygens within the cavity; (2) the hydrogen-bonding interactions between the secondary hydroxyl groups at the edges of [3-CD and the functional groups attached to the t-butyl moiety; and (3) the hydrogen bonding between the functional groups attached to the t-butyl moiety and the bulk (methanol/water) solution. The magnitude of the observed modifier effects would, therefore, depend on how these forces counterbalance, which, in turn, depends on the chemical structure of the substituent groups attached to the modifier.

The binding to [3-CD may involve only a single guest, as in binary [3-CD complexes, or two molecules, as in the formation of guest/modifier/[~-CD ternary complexes. One could actually predict from the foregoing discussion that a nonpolar guest molecule with appropriate polar substituent(s) with which it can effectively hydrogen bond to the hydroxyl groups at the edges of a given CD and to the solution medium, would form more stable complexes with [3-CD than would one without such polar substituent(s). Such a system would exhibit little or no concomitant effects. To illustrate this point, we conducted a fluorescence study on the concomitant effects of cyclo-

1 , 4 0

1 . 3 0

1 . 2 0

1 . 1 0

1.OO

0 . 9 0 ~ t 1.0 2 .8 4 . 6 6 . 4 8 .2 1 0 . 0

pH

FIe. 5. Influence of pH on the VIII ratio of pyrene in the presence of 0.075 M of various t-butyl modifiers: blank (+); TB-alcohol (V); TB- acetate (1); TB-carbazate (O); and TB-carbamate ([]).

pentanol, t-butyl carbamate, and t-butyl carbazate on the formation of [3-CD/8-hydroxyquinoline and [3-CD/quinoline complexes. The results strongly suggest that the presence of any one of these modifiers has no significant effect on the formation of their [3-CD/8-hydroxyquinoline complexes but does have an effect on the [3-CD/naphthalene system. More work is required in this area before any conclusive statement can be made regarding the enhancement of [3-CD/guest complexes by appropriately substi- tuting the guest.

T h e Ef fec t s o f V a r y i n g the C o n c e n t r a t i o n o f the M o d i f i e r . The effects on the I/III ratio of varying the concentration of the most active TB-compounds (Fig. 2) from 0 to 1.0 M were examined, and the results are plotted in Fig. 4. As can be seen, the VIII ratio values de- crease dramatically and reach a minimum around a concentration of 0.010 M for most modifiers.

I n f l u e n c e o f p H o n the Ef fec t s o f M o d i f i e r s . The modifier compounds tested contain a variety of functional groups in their structure; some are acidic, others basic, and the remainder are neutral. One would, therefore, think that their effects could be pH-dependent. Solutions of pH values around 2.0, 6.0, 7.5, and 9.0 were prepared in 60% methanol/40% water solution as described in Refs. 21 and 22. The final pH values of these solutions, containing 0.075 M of the active modifiers, 1.0 × 10 : M pyrene, and 3.0 mM ~-CD, were checked with an appropriately calibrated pH meter. The indeterminate potential at the junction of the alcohol-solution with the use of standard pH meter makes the interpretation of these pH values very difficult. 2~,22

Plots of I/III ratio vs. pH for TB-alcohol, TB-acetate, TB-carbamate, and blank (methanol/water), in 3 mM [3-CD, are shown in Fig. 5. These plots show no significant change in the I/III ratios. However, the relative values of the I/III ratios in these solutions, which range from 1.34 in methanol/water blank to 1.03 in TB-carbamate, suggest various levels of the [3-CD/pyrene/modifier equi- libria under these conditions. For example, the TB-carbamate shows the lowest I/III ratio value, which corre- sponds to the strongest effect, in keeping with Fig. 2. This result would be expected if the functional groups on the t-butyl moiety were not encapsulated by [3-CD and

998 Volume 50, Number 8, 1996

3.30

o L~

T o

O

o

o

2.64

1,98

1,32 i

0.66

/ \ /

/ ,~\\\ f / / . / / ~ \ / / / , \ /

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Wave leng th , nm

FIG. 6. Emission spectra of pyrene in the presence of 0.075 M TB- carbamate at pH 2 (bottom); pH 6 (top), and pH 9 (middle).

were far removed from pyrene in the ternary structure so as not to affect its electronic structure. Emission spectra of pyrene in 0.075 M TB-carbamate and 3 mM [3-CD at pH 2, 6, and 9 are shown in Fig. 6. Some shifts in in- tensity are observed, but there is no significant change in the I/III ra t io--which, again, implies that there is no signif icant change in the concen t ra t ion of the ~-CD/ pyrene/modifier complex under these different pH conditions. This observation is in keeping with the obser- vations from the study with reversed-phase liquid chro- matography. 2° The effects of pH on the I/III ratio of pyrene in the presence of TB-formate and N-(t-butoxycarbonyl) glycine are not reported, because their solutions were acidic under all conditions.

C O N C L U S I O N

In this study, an attempt has been made to examine the ef fec ts of t -butyl compounds on the fo rmat ion of ~-CD/pyrene complexes in a solution medium comprised of 60% methanol in water. It was believed that varying the functionality on the t-butyl moiety could, perhaps, shed some light on the mechanism(s) of the formation of the [3-CD/pyrene/TB-modifier ternary complexes. The results show that the stability of the ternary complex, in- deed, depends on the functionality on the t-butyl moiety. For example, the observed effects for the TB-carbamate and TB-carbazate are significantly higher than the effects for TB-alcohol under similar solution conditions. Appar- ently the additional hydrogen-bonding sites in the R group in the general structure, (CH3)3-C-O-R, makes a big difference in the stability of the ternary complex. The presence of the - C - O - , bridge in the general structure of the modifier apparently is necessary for strong interaction (possibly of hydrogen-bonding type) between the modifier molecule and the secondary hydroxyl groups at the edges of [3-CD. Lack of the - C - O - bridge in the general

structure results in minimal or no effects, as observed with TB-amine, TB-acetic acid, and TB-acetoacetate. Ob- viously, the magnitude of the observed modifier effects is determined by how well these forces counterbalance, which, in turn, depends on the modifier chemical structure. Further studies will involve closer examination of the hydrogen-bonding effects by NMR and other tech- niques. Potential applications of the modifier effects in- clude selective inclusion and subsequent separation or extraction of ternary complexes in complex matrices and improved solubility of [3-CD in solutions of organic-water mixtures.

A C K N O W L E D G M E N T

This work was supported by the National Science Foundation (CHE- 9224177). The authors are grateful to G.A. Reed of American Maize Products for providing the [3-CD samples used in this study.

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2. M. Kurihara, E Hirayama, K. Uekama, and M. Yamasaki, J. Incl. Phenom. 8, 363 (1990).

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Pharm. Bull. 26, 3477 (1978). 13. G. Patonay, K. Fowler, G. Nelson, and I. M. Warner, Anal. Chim.

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Live, and I. M. Warner, J. Am. Chem. Soc. 133, 1572 (1991). 16. A. Mufioz de la Pefia, T. T. Ndou, J. B. Zung, and I. M. Warner, J.

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(1988).

APPLIED SPECTROSCOPY 999

fluorescence studies of the effects of t-butyl functionalities on the formation of ternary...

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