Enhanced Room-Temperature Phosphorescence of Anthracene on Cyclodextrin-Treated Filter Paper

Enhanced Room-Temperature Phosphorescence of Anthracene on Cyclodextrin-Treated Filter Paper*

T U A N V O - D I N H t and ALA ALAK Advanced Monitoring Development Group, Health and Sa[ety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6101

This study investigates the enhancement of room-temperature phosphorescence (RTP) for anthracene when the compound is adsorbed on cyclodextrin (CD)-treated filter paper. The results show that/~-CD treatment induces a significant increase in anthracene RTP emission, which is normally extremely weak. The a-CD does not produce any strong RTP enhancement, while 3'-CD produces relatively lower enhancement than does fl-CD. The CD treatment procedure is very simple and improves the analytical usefulness of the RTP method. The binding constants of anthracene to cyclodextrin were measured with the use of thin-layer chromatographic retention values. Anthracene has a higher binding constant with fl-CD than with "y-CD, in agreement with the RTP enhancement results.

Index Headings: Room-temperature phosphorescence; Anthracene; Cy- clodextrin; Organic analysis.

INTRODUCTION

Since the first report by Roth, less than two decades ago, 1 room-temperature phosphorimetry (RTP) has be- come a powerful technique for a wide variety of applications. ~ The RTP technique, unlike conventional low- temperature phosphorimetry (LTP), is characterized by the simplicity of its experimental procedures. For the past few years, RTP has stimulated greater interest among analytical chemists than has the older LTP technique. The popularity of RTP is mainly due to versatility and the applicability of this technique to many types of solid substrates, such as filter paper, '~-s silica gel, 7 inorganic materials, s poly (acrylic acid)-based mixtures, 9 and liquid samples, l°,u Recently, RTP has been reported for liquid samples containing cyclodextrin, t2 14 Cyclodextrin-NaC1 mixtures were also reported to induce RTP from a variety of organic compoundsJ ~ Alak et al. TM studied the effect of cyclodextrin and surfactant reagents on the fluorescence densitometry of polycyclic aromatic hydrocarbons and dansylated amino acids. They observed fluorescence enhancement with the use of the cyclodextrin or surfactant sprays when silica gel and alumina thin-layer chromatography (TLC) plates were used, but either no effect or a slight decrease in fluorescence when reverse-phase cellulose or polyamide plates were used. In this work, we investigate the usefulness of a simple substrate based on filter paper treated with cyclodextrin for RTP analysis.

Cyclodextrins (CD) are natural macrocyclic polymers of glucose that contain from six to twelve D-(+) gluco- pyranose units. 17-'~° The cyclodextrins are not cylindrical,

Received 13 February i987. * Research sponsored by the Office of Health and Environmental Re-

search, U.S. Department of Energy, under Contract DE-AC05- 840R21400 with Martin Marietta Energy Systems, Inc.

t Author to whom correspondence should be sent.

but have the shape of a toroid or hollow truncated cone. Although cyclodextrins containing as many as twelve glucose units have been identified, only cyclodextrins con- sisting of six, seven, and eight glucose units are most commonly used; these cyclodextrins are commonly called a, fl, and ~/-cyclodextrin, respectively. 17 20

The ability of cyclodextrins to form inclusion complexes with a variety of compounds should make cyclodextrins ideal systems to affect photophysical processes such as RTP. The relative stabilities of cyclodextrin inclusion complexes are governed by factors such as hy- drogen bonding, hydrophobic interaction, and solvation effects, as well as the size of the guest molecule. The unique ability of cyclodextrin to form inclusion complexes with other compounds has been advantageously utilized in many agricultural, pharmaceutical, industrial, and analytical applications. 21 25

In this study, we have selected anthracene as the model compound to investigate the effect of cyclodextrins on RTP using cellulose substrates. Anthracene is a partic- ularly important compound for RTP analysis, since this compound is commonly found in environmental samples such as wastewaters and air sample extracts and is on the U.S. Environmental Protection Agency Priority Pol- lutant List. The phosphorescence intensity of anthracene, however, is very weak and thus has not been extensively investigated by RTP. ~ In this study, we investigate the phosphorescence enhancement induced by the treatment of filter paper substrate by cyclodextrins. The different effects of a-,/3-, and 7-cyclodextrins on RTP are discussed in detail. Also, the binding constants of anthracene to a-, fl-, and -y-cyclodextrin molecules were measured with the use of TLC techniques, with cyclodextrin as the mobile phase. The results indicate that the strength of the binding constant of anthracene can be correlated with RTP enhancement.

EXPERIMENTAL

Apparatus. All spectra were obtained with a Perkin- Elmer spectrofluorimeter (Model 43A, Perkin-Elmer, Norwalk, CT) with a phosphoroscope attachment. A 150-W xenon-arc lamp was used for excitation. The de- tector was a photomultiplier (Hamamatsu Co., Mid- dlesex, NJ; Model R-777) with a photocathode spectral response from 185 to 850 nm. The spectra were not cor- rected for instrument response. Special laboratory-con- structed sample holders were used for RTP measure- m e a t s . 26

Reagents. Anthracene was purchased from Aldrich Chemical (Milwaukee, WI) and used as received, without

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further purification. The solvent used in the preparation of all samples was ethanol of spectroscopic grade (Aaper Alcohol and Chemical Co.). The heavy-atom salts for RTP analysis were commercially available and used in ethanol-water mixtures (volume ratio 1:1). Thallium acetate was purchased from Fluka Chemical Co. (Haup- pange, NY), and lead acetate from Aldrich Chemical Co. Cyclodextrin (~, fl, and ~) was purchased from Al- drich and used as received. Whatman filter paper (What- man 1) was used as the RTP sample substrate.

Procedure. Whatman filter papers were cut into 1-cm- diameter disks and impregnated in saturated aqueous solutions of a-, fi-, and ~-cyclodextrinfor two hours. The filter papers were removed from the cyclodextrin solutions and allowed to dry in ambient air. The RTP measurements were conducted according to the procedure described previously. 26

The binding constants of anthracene to cyclodextrin molecules were measured by the TLC method. All TLC developments were conducted with the use of a 3-in-high, 1-in-diameter developing chamber. Cyclodextrin mobile- phase concentrations were 0.05, 0.04, 0.03, 0.02, and 0.01 M. All solutes were developed with the use of high-per- formance liquid chromatography (HPLC) grade water as the mobile phase. Polygram polyamide-6 TLC plates (Macherey-Nagel, West Germany) were used for all de- terminations. All measurements used 1-t~L samples of 10 3 M anthracene solutions. The TLC spots were vi- sualized by fluorescence quenching.

RESULTS AND DISCUSSION

Figure 1 shows RTP spectra of anthracene adsorbed on untreated filter paper (Fig. la) and filter paper substrates treated with CD (Fig. lb, lc, and ld). All samples contained the same amount of anthracene (53.4 ng) and were analyzed on filter paper treated with thallous acetate as the heavy-atom agent. Without the heavy-atom agent, no RTP would be observed, either with or without cyclodextrin. The results showed that anthracene RTP emission was barely distinguishable at approximately 674 nm on the substrate with no cyclodextrin (Fig. la) and on the substrate treated with a-CD (Fig. lb). The broad band from 500 to 640 nm corresponds to the substrate background emission. The a-CD molecules, which con- sist of six glucose units, have a cavity internal diameter of approximately 5.7/~8 and thus are not large enough to trap the anthracene molecule. When filter paper was treated with fl- and ~/-CD, which have cavity internal diameters of approximately 7.8 and 9.5/~,~s respectively, inclusion complexes with anthracene could be formed and RTP emission of anthracene could be clearly detected (Fig. lc and ld). The RTP spectra were charac- teristic of anthracene, exhibiting an intense 0,0 band at approximately 674 nm, a weak shoulder at ~ 690 nm, and another peak at ~ 744 nm which corresponds to the 1400- cm 1 C-C vibronic band of polyacenes. A small batho- chromic shift (~4 nm) was observed for the 0,0 band of anthracene on/~-CD- and -y-CD-treated paper. It was the first time, to the best of our knowledge, that reasonably strong RTP emission of anthracene on solid substrate was reported. Although polycyclic aromatic hydrocarbons have been extensively investigated with the use of

3

2

1

3

~ 2 r-

~ 5 <

>. 4

z 3 W

z 2

n -

RTP OF ANTHRACENE, EXC. WAVELNGTH = 375 nrn

b) WITH ALPHA-CD

I I I I I , I

d) WITH GAMMA-CD

520 600 680 760

WAVELENGTH (nm)

Fro. 1. RTP emission spectra of anthracene adsorbed: (a) on filter paper without cyclodextrin (CD); (b) on filter paper t rea ted with a-CD; (c) on filter paper t reated with j3-CD; (d) on filter paper t rea ted with -y-CD.

RTP, and many of them exhibited strong RTP emission when an external heavy-atom perturber was used, anthracene has always been reported to show no, or extremely weak, phosphorescence emission. We have previously studied anthracene RTP and reported that RTP was not analytically useful for anthracene, since samples spotted from a saturated solution of anthracene in ethanol could barely be detected, even with silver nitrate as the heavy atom agent. 2 The results of this study dra- matically demonstrate that a simple treatment of filter paper with/~- and ~-CD provides an efficient procedure for improving RTP detection of anthracene.

Among polynuclear aromatic hydrocarbons, anthracene represents an interesting particular case. Previous studies of the energy levels, 27-3° the dependence of the t r iple t quan tum yield on temperature , ~1 and laser emission ~2 suggested the location of a second triplet state T2 (or possibly T3) in the vicinity of the lowest excited singlet state S~. This situation of near degeneracy of T2 and S~ appears to be unique among polycyclic aromatic hydrocarbons and induces a strong dependency of the intersystem crossing of anthracene on matrix or temperature effects. Since our results of RTP measurements with/~-CD and ~-CD show strong phosphorescence, it is of interest to study whether there was any spectral shift

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3 RTP OF ANTHRACENE, EM. WAVELENGTH = 678 nm a) NO CD

2

I I i I i I I

3 b) WITH ALPHA-CD

>. 4

z 3 W k- z 2

5 d) WITH GAMMA-CD

3

2

1

1 I i i i i i

260 340 420 500

WAVELENGTH (nm)

FIG. 2. R T P excitat ion spec t ra of an th racene adsorbed: (a) on filter paper wi thout cyclodextr in (CD); (b) on filter paper t rea ted with a-CD; (c) on filter paper t rea ted with #-CD; (d) on filter paper t rea ted with y-CD.

in the singlet and/or triplet states induced by the use of CD-treated substrates. The RTP spectra of anthracene in paper substrate with no CD and with a-CD were too weak to compare with the triplet states of anthracene in #-CD and ~-CD. Phosphorescence excitation spectra were performed to compare the singlet state energy levels of anthracene adsorbed on various substrates. The results shown in Fig. 2 indicate that the single state S~ of anthracene was blue-shifted by about 5 nm when/~-CD and ~/-CD were used (Fig. 2c and 2d). These shifts occur when the strongest phosphorescence enhancement is observed, suggesting that a slight change in the S~ energy might affect the intersystem crossing process because of the near degeneracy of the S~ and T~ levels. This effect, in conjunction with the molecular inclusion process previously discussed, could cause an increase in intersystem crossing, leading to increased RTP emission.

Binding Constants Studies. Armstrong et al. developed an efficient thin-layer chromatographic method to determine the binding constants of different organic compounds with a-CD. 33,34 In this study, the method is applied to other cyclodextrin molecules (#- and 7-CD) in order to investigate the binding constant of polynuclear aromatic hydrocarbons to cyclodextrins.

Equations were originally derived to determine a-cyclodextrin-substrate complex stoichiometric parameters as well as primary and secondary binding constants with the use of TLC technique. One of the equations is:

1 Rf kl[CD] klk2[CD] 2 + ( I )

k' 1 - Rf Ok[A] Ok[A]

where k' is the capacity factor; Rf is the retardation factor of a solute in thin-layer chromatography (a parameter denoting the ratio of the distance traveled by the mobile phase over the distance traveled by the solute); k is the equilibrium constant between the solute and the sta- tionary phase; and kl and k2 are the equilibrium constants for the substrate and cyclodextrin (kl for a 1:1 solute : cyclodextrin complex, k2 for a 1:2 solute: cyclodextrin complex). When the cyclodextrin-solute stoichiometry is one to one (k2 = 0), straight line plots are obtained. In this anthracene study the stoichiometry was 1:1 for/~- and 7-cyclodextrin. • is the phase ratio, [CD] is the concentration of cyclodextrin, and [A] is the concentration of the solute.

1 Rf Plots of ~ or 1 - R-----~ vs. cyclodextrin concentration

give a straight line in which the slope corresponds to kl 1

Ok[A----] and the intercept Ok[A-----l" From the ratio of the

slope over the intercept values one can calculate kl. Val- ues for kl for anthracene were 32.39 and 9.41 for/~- and 7-cyclodextrins, respectively. Rf values of zero were obtained for a-CD at all concentrations investigated in this study. The Rf values of zero indicate that the binding constants of anthracene to a-CD are zero and explain why no RTP enhancement was observed when we used a-CD. The binding constant values indicate that inclusion of the solutes with the cyclodextrin molecules is an important factor for achievement of any enhancement in RTP signal. The ~-cyclodextrin, which has the largest kl value (32.39), produces the best enhancement for anthracene RTP. The ~-CD, which has a weaker binding constant (9.41) with anthracene than does fl-CD, shows less enhancement for RTP than does fl-CD.

CONCLUSIONS

Room-temperature phosphorimetry is a practical tool for chemical analysis. This study shows that treating filter paper with CD can further enhance the sensitivity of this analytical technique. The procedure is simple and does not require any chemical treatment. Also, the RTP measurements with CD-treated filter paper do not require removal of oxygen from the samples, as in liquid samples. The binding of the analyte molecules to the cyclodextrin molecules appears to be an important factor in producing RTP enhancement. The TLC method provides an efficient and effective way to determine the binding constants of analyte/cyclodextrin systems. The applicability of CD-treated substrates to other polycyclic hydrocarbons is currently under investigation in this laboratory.

APPLIED SPECTROSCOPY 965

ACKNOWLEDGMENTS

This research is sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05- 840R21400 with Martin Marietta Energy Systems, Inc.

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and K. Bui, Anal. Chem. 57, 234 (1985). 25. A. Alak and D. W. Armstrong, Anal. Chem. 58, 582 (1986). 26. T. Vo-Dinh and P. R. Martinez, Anal. Chim. Acta 13, 125 (1981). 27. S. P. McGlynn, T. Azumi, and M. Kinoshita, Molecular Spectros-

copy of the Triplet State (Prentice-Hall, Englewood Cliffs, New Jersey, 1969).

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Investigation of Experimental Parameters for Surface-Enhanced Raman Scattering (SERS) Using Silver-Coated Microsphere Substrates*

R. L. MOODY, t T. VO-DINH,~ and W. H. FLETCHER:[: Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (R.L.M., T.V.-D.); and Chemistry Department, University of Tennessee, Knoxville, Tennessee 37919 (W.H.F.)

The effects of experimental parameters on surface-enhanced Raman scattering (SERS) for silver-coated microsphere substrates were investigated. The parameters included sphere size, silver thickness, and excitation wavelength. The SERS signal intensity varied as each of these parameters was changed. The potential for increasing SERS sensitivity was illustrated by the detection of benzo(a)pyrene, a molecule of environmental and toxicological interest, at a concentration well below that possible for normal Raman scattering. This work describes some opti- mization procedures and demonstrates the possibility for further im-

Received 13 March 1987. * Research sponsored by the Department of the Army under Inter-

agency Agreement Numbers DOE 40-1294-82 and Army 3311-1450, and the Office of the Health and Environmental Research, U.S. De- partment of Energy, under Contract DE-AC05-84OR21400 with Mar- tin Marietta Energy Systems, Inc.

t Also a graduate student at The University of Tennessee, Knoxville. Authors to whom correspondence should be sent.

proving SERS enhancement of these silver-coated microsphere substrates by optimizing experimental parameters.

Index Headings: Raman; Laser; Analysis; Detection; Surface-enhanced Raman scattering.

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

S u r f a c e - e n h a n c e d R a m a n s ca t t e r i ng ( S E R S ) has b e e n an ac t ive a rea of r e sea rch s ince its d i scove ry in 19741 a n d c o n f i r m a t i o n in 1 9 7 7 Y M o s t of t he r e sea rch to d a t e has b e e n d i r e c t e d t ow ards f u n d a m e n t a l s tud ies of m e c h a - n i sms r e spons ib l e for t h e e n h a n c e m e n t . 4,~ A l t h o u g h t h e r e are a variety of theoretical models, the theories which have evolved may be divided into two major groups.

The first group attributes the enhancement to modification of the molecular polarizability and hence the Raman cross section. Modification of the molecular polarizability is caused by interaction of the molecule and

966 Volume 41, Number 6, 1987 0003-7028/87/4106-096652.00/0 APPLIED SPECTROSCOPY © 1987 Society for Applied Spectroscopy

Enhanced Room-Temperature Phosphorescence of Anthracene on Cyclodextrin-Treated Filter Paper

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