2024
Anal. Chem. 1991, 63, 2924-2933 Peacocke. A. R.; Richards, R. E.; Sheard, B. Mol. Phys. W68, 76, 177. Cohn, M.; Townsend, J. Nature 1954, 773, 1090. Cooper, M. D.; Winter, P. K. Treatise on Andytkal Chemistry; John Wiiey 8 Sons, Inc.: Easton, PA, 1961; Part 11. Voi. 7, p 425. Brown, R. D., 111; Brewer, C. F.: Koenig, S.H. Blochemishy 1977, 76. 3883. Brewer, C. F.; Brown, R. D.. 111: Koenig, S. H. Biochemistty 1983, 22. 3691. Kang, Y. S.; Gore, J. C.: Armitage. I.M. Magn. Reson. Med. 1984, 7 , 396. Koenig, S. H.; Bagiin, C.; Brown, R. D., 111. Magn. Reson. Med. 1984, 7 , 496. Koenig, S. H.; Brown, R. D., 111. Magn. Reson. Med. 1984, 7 , 478. King, J.; Davidson, N. J. Chem. Phys. 1958, 29, 787. Koenig, S. H.; Brown, R. D., 111. J. Magn. Reson. 1985, 67,426. Koenig, S. H.; Brown, R . D., 111. Studebaker, J. CoM Spring Harbor Quant. Biol. 1971, 36, 551. Koenig, S. H.; Brown, R. D., 111; Spiller, M. Magn. Reson. Med. 1987, 4 , 252. Keiiar, K. E.: Spaltro. S. M.; Foster, N. Macromolecules 1990, 2 3 , 428. Koenig, S. H.;Brown, R. D.,111. I n NMR Spectroscopy of Cells and &@nisms; Gupta, R. K., Ed.; CRC Press Inc.: B o a Raton. FL, 1987. Maddams, W. F. Appl. Spectrosc. 1960, 3 4 , 245. Martin, M. L.: Depuich, J.J.; Martin, G. J. Practical NMR Spectrosco-
py: Heyden 8 Son, Ltd.: London, U.K., 1980. (19) Bernheim, R. A.; Brown, T. H.; Gutowsky, H. S.; Woessner, D. E. J. Chem. P h p . 1959, 3 0 , 950. (20) Bioembergen, N.; Morgan, L. 0. J . Chem. phvs. 1961, 3 4 , 1961. (21) Koenig, S. H.; Brown. R. D., 111; Kurland, R.; Ohki, S. Magn. Reson. Med.%& 7 , 133. (22) Lyon, R. C.; Faustino, P. J.; Cohen, J. S.; Katz, A.; Mornex, F.; Cotcher, D.; Baglin, C.; Koenig, S. H.; Hambright, P. Magn. Reson. Med. 1987. 4 . 24. (23) Geralbes,C. F. G. C.; Sherry, A. D.; Brown, R. D., 111; Koenig. S. H. h4agn. Reson. Med. 1986, 3 , 242. (24) Krishnamurthy, M.; Sutter, J. R.; Hambright, P. J. Chem. Soc., Chem. Common. 1975. 13. (25) Pasternack, R. F.; Huber, P. R.; Boyd, P.; Engasser, G.; Francesconi, L.; Gibbs, E.; Fasella, P.; Cerio Venturo, G.; Hinds, L. deC. J. Am. Chem. Soc. 1972, 94, 4511. (26) Melton, B. F.; Pollak, V. L. J. Phys. Chem. 1969, 73, 3669.
RECEIVED for review May 15,1991. Accepted September 23, 1991. This work was supported by grants from the American Cancer Society, P D T No. 327C (N.F.), and the National Institute of General Medical Sciences, USPHS No. GM44439
(N.F.).
Evaluation of the Relative Effectiveness of Different Water-Soluble @-CyclodextrinMedia To Function as Fluorescence Enhancement Agents Raymond P. Frankewich,
K.N. Thimmaiah, a n d Willie L. Hinze*
Department of Chemistry, Laboratory f o r Analytical Micellar Chemistry, Wake Forest University, P.O.Box 7486, Winston-Salem, North Carolina 27109
The effects of @-cyclodextrlnas well as four water-soluble 8-cyclodextrln systems, 1.e. urea-solublllred B-cyclodextrln, and the water-soluble derlvatlves, hydroxyethyl-B-cyclodextrin, 2-hydroxypropyl-~-cyclodextrln,and heptakls (2,6dl-0-methyl)-&cyclodextrln, upon the fluorescence behavior of 14 dansylamlno aclds and 33 organlc/pharmaceutlcal compounds were determlned. I n addltlon, fluorescence data on these solutes were obtained In the homogeneous solvents water and methanol. The use of the more water-soluble 8cyclodextrin systems typlcally resulted In greater fluorescence from these 47 compounds compared to that obtainable with natlve 8-cyclodextrln. I t Is thought that the added fluorescence enhancements observed are due to the fact that a greater fractlon of the solute molecules are complexed within the protectlve cyclodextrln cavlty at the greater cyclodextrln concentratlonsobtalnable wRh these water-soluble systems. The use of elther 2-hydroxypropyl- or 2,6-dl-O-methyl-@cyclodextrln generally resulted In the greatest fluorescence enhancement factors and Increased the stablllty of some fluorescent systems. The former 8-cyclodextrln derlvatlve offers an advantage compared to the rest In terms of ease of volume handllng/manlpulatlon conslderatlons due to Rs lower solutlon viscosity. The resuRs Indicate that If a &cyclodextrln-enhanced fluorescence assay Is belng contemplated, use of 2-hydroxypropyC~-cyclodextrlnIn lleu of natlve &cyclodextrln should be consldered.
INTRODUCTION Cyclodextrins are cyclic oligosaccharides composed of six or more glucopyranose moieties bonded together via 1,4-ether
linkages. The three most commonly employed cyclodextrins are a,p, and y, which contain six, seven, and eight of these glucopyranose units, respectively. The structure of the cyclodextrins is that of a truncated cone with average cavity diameters of 0.57, 0.78, and 0.95 nm for a-,p-, and y C D , respectively. The apolar cavity is lined by the glucopyranose carbon and hydrogen atoms and glycosidic oxygens. The more open edge of the cyclodextrin is lined with secondary hydroxyl groups (two per glucopyranose unit) while the opposite edge has primary hydroxyl groups (one per glucopyranose unit). Solute molecules of the correct dimensions can interact and bind to the CD cavity via inclusion complex formation. The reduced polarity and restricted microenvironment provided by the CD cavity can markedly and advantageously influence a number of properties of an included solute (1, 2). As a consequence, cyclodextrins have been increasingly utilized in the past decade as additives in pharmaceuticals, foods, cosmetics, pesticides, etc. and as reagents in analytical chemistry (for monographs on cyclodextrins, refer to refs 1-5). Considerable attention has been focused on the use of cyclodextrins in luminescence applications (6-14). This stems from the fact that, upon complexation with cyclodextrins, many analyte molecules exhibit enhanced luminescence efficiencies compared to that observed in bulk water. The factors thought to be responsible for such intensified luminescence include shielding of the cyclodextrin-complexed analyte molecule from quenching by water molecules or solvent-borne quenchers and increased local viscosity in the CD cavity with a concomitant reduction of oxygen quenching. In addition, the included solute molecule experiences a less polar and more rigid local microenvironment (4,13). The polarity of the CD cavity has been estimated to be similar to that of oxygenated solvents such as dioxane, ethanol, 1-octanol, iso-
0003-2700/91/0363-2924$02.50/0@ 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
propyl ether, ethylene glycol, or tert-amyl alcohol (13, 15-18). As a consequence, the cyclodextrin cavity can protect analyte excited states from nonradiative and quenching processes that normally readily occur in bulk aqueous solution. In essence, cyclodextrin complexation of analyte molecules results in a sort of "poor man's'' matrix isolation procedure for luminescence assays. The presence of cyclodextrins can dramaticaly enhance the fluorescence signal of complexed solutes (19-36). The first application of cyclodextrins in this regard appears to have been reported by Kinoshita and co-workers for dansylated amino acids in a series of papers in the mid 70s (6, 19-24). Fluorescence enhancements have since been observed for a wide variety of other analyte molecules. Due to its intermediate cavity size which allows for complexation with a greater array of analyte molecules, 0-cyclodextrin (P-CD) has been more generally utilized for such fluorescence applications. However, in many instances, it has been observed that the magnitude of the fluorescence enhancement attainable with 0-CD is restricted due to its limited water solubility (ca. 0.0144.016 M). That is, the fluorescence intensity of many analyte molecules increases with cyclodextrin concentration until the p-CD solubility limit in water is reached (6, 19, 27-32). Since the analyte-CD complexation process is a dynamic equilibrium situation (eq l),the analyte fluorescence analyte
+ p-CD Kb. ana1yte.P-CD complex
(1)
dependence upon cyclodextrin concentration is no doubt due to the increased proportion of analyte molecule which is included in the protective CD cavity. The implication is that the fluorescence intensity of many analytes should be still further intensified if it were somehow possible to increase the 0-cyclodextrin solution concentration further above its water solubility limit. In this paper, we compare the contrast the general properties and ability of different water-soluble P-cyclodextrin systems to function as enhancement agents in fluorometric analysis. The enhanced water-soluble P-cyclodextrin media examined include urea-solubilized P-CD (urea/p-CD) as well as some water-soluble derivatized P-CDs; i.e., 2-hydroxypropyl-P-CD (HP-0-CD), hydroxyethyl-P-CD (HE-P-CD),and 2,6-di-O-methyl-P-CD (DOM-P-CD). Data were also obtained in the homogeneous solvents, water and methanol, and in aqueous native P-CD solutions. The results, based upon examination of a large number of test analytes, indicate that use of HP-P-CD or DOM-P-CD typically yielded the greatest enhancements in luminescence intensity and improvements in analyte sensitivity. Their use in lieu of native p-CD is recommended for analytical luminescence measurements. EXPERIMENTAL SECTION
Instrumentation. All fluorescence spectra and intensity measurements were made on an American Instruments Aminco-Bowman spectrophotofluorometer (equipped with a 1P21 PM tube, S-4 response and Houston Instruments chart recorder). Typically, the excitation, emission, and detector slit widths were set at 1 mm (which corresponds to a 5.5 nm band-pass). Absorbance spectra were measured on a Varian Cary 219 UV-visible spectrophotometer. Viscosity measurements were carried out at room temperature with a calibrated Ostwald viscometer. Surface tension measurements were made using a Fisher Model 20 tensiometer fitted with a platinum ring (de Nouy ring method). All pH measurements were made on a Corning Model 135 pH/ion meter. Mass spectral data on the 2-hydroxypropyl-~-cyclodextrin derivative (provided by the commercial supplier, Pharmatec, Inc.) were obtained by use of negative-ion fast atom bombardment mass spectrometry. The average molecular weight and degree of substitution (DS) of the derivatized cyclodextrins can be determined from such mass spectrometry data. The degree of sub-
2025
stitution can be calculated from eq 2, where NS refers to the number of substituents (37, 38). DS = (peak height X NS)/(peak height) (2) Materials. @-Cyclodextrin(P-CD) and hydroxyethyl-Pcyclodextrin (HE-p-CD) (Advanced Separation Technologies, Whippany, NJ), an aqueous 45% (w/v) concentrated stock solution (Batch No. 88-02-505-1)or solid (Batch No. 88-02-506-1) of Molecusol, i.e. 2-hydroxypropyl-~-cyclodextrin (HP-P-CD) (Pharmatec, Inc., Alachua, FL), heptakis (2,6-di-O-methyl)-Pcyclodextrin (DOM-P-CD) (Aldrich, Milwaukee, WI), and urea (Calbiochem, San Diego, CA, or Fisher Scientific Co., Raleigh, NC) were used as obtained without further purification. The analytes,dansyl amino acids (Sigma Chemical Co., St. Louis, MO, or Pierce, Rockford, IL), indole and salicyclic acid (Fisher), 1naphthol, 5-methoxypsoralen,nystatin, fenproporex, furosemide, bombesin, retinol, retinal, chlortetracycline,diazepam, prazepam, d,l-a-tocopherol, and procaine hydrochloride (Sigma),quinizarin, 4-amino-N-methylphthalimide, diphenyl phosphate, aflatoxin B1, DAPI, N-methylaniline, 1,8-dihydroxyanthraquinone,methyl salicylate, 2-naphthol, pamoic acid, and 4-(N,N-dimethylamin0)benzonitrile (Aldrich), 4-aminophthalimide, erythrosin, N-phenyl-1-naphthylamine, coumarin I, and merocyanine 540 (Kodak, Rochester, NY), and p-aminobenzoic acid (Merck, Rahway, NJ) were all used as obtained. The dansyl derivatives of phenolic steroids were prepared by reaction of dansyl chloride with the steroid using a literature procedure (39). 2-Methylhydronaphthoquinone-1,4 was prepared via the reduction of menadione with stannous chloride using a previously reported procedure (40). The solvents utilized included water (Fisher HPLC grade or in-house purified (Milli-Q Reagent Water System (Millipore Corp.)), DMSO (Sigma or Aldrich), methanol (Fisher HPLC grade), acetonitrile (Fisher HPLC grade), and deuterium oxide (Aldrich gold label). The buffers employed for pH adjustment included Fisher certified pH 6.0, 7.0, or 8.0 standard buffer solutions (0.05 M phosphate systems). All other chemicals and solvents were of the best grade commercially available and were used without further purification. Procedures. Cloud Point Determinations. The cloud point behavior of the different CD media was determined by monitoring the temperature at which a particular aqueous cyclodextrin solution turned turbid upon heating or clear again upon subsequent cooling. In all measurements, the highest temperature employed was ca. 100 "C. Fluorescence Measurements. Typically, stock solutions of the different analytes were prepared by dissolving 2-5 mg of the solid material in 10 mL of solvent (water,methanol, DMSO, or dioxane plus, in some cases, appropriate buffer). These solutions were usually freshly prepared just prior to use. The aqueous stock solutions of the different cyclodextrin media were prepared by weighing out the appropriate amount of the solid cyclodextrin material and diluting to final volume with distilled water (or aqueous urea solution to achieve more concentrated solutions of native 0-cyclodextrin) or by the appropriate dilution of a concentrated 45% (w/v) source stock solution in the case of HP-P-CD. For the spectrofluorometric measurements in the different homogeneous solvent systems (water, methanol, or acetonitrile) or cyclodextrin media, the relative fluorescence intensity of the analyte was typically measured by employing at least two different analyte concentrations. These solutions were prepared by injection of 20 (or 10) and 40 (or 20) pL of the concentrated analyte stock solution into 10.00 (or 5.00) mL of the homogeneous solvent or aqueous cyclodextrin system under study. All fluorescence intensity measurements have been corrected for the background due to the medium alone by use of appropriate blanks. The respective fluorescence excitation and emission wavelengths employed are listed in the data tables. In order to avoid hydrolysis of some of the analytes (e.g. methyl salicylate, retinal), the luminescence measurements were conducted within 1h after their preparation. Determination of Analyte-Cyclodextrin Binding Constants. Binding constants in the different CD media were determined using either the fluorescence or thin-layer chromatographic method. In the fluorescence procedure, equilibrium binding constants were determined by measuring the fluorescence intensity of the
2026
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
Table I. Summary of Physicochemical Properties of 8-Cyclodextrin and Selected Solubilized 8-Cyclodextrin Systemsa
B-CD-containing medium native j%CD Re = R2 = R3 = H urea-solubilized R6 = Rz = R3 = H native 0-CD HE-8-CD replacement of R's = H by one or more -CH2CHz0H moieties HP-8-CD replacement of R's = H with one or more -CH2CH(OH)CH3 moieties Re = R2 = CH3; R3 = H DOM-8-CD
solubilitv av molar av in watei substi- molecular at 25.0 tutionc mass" OC, M 1135 1135
viscosity (at 23 "C), CP
0.01638 0.982 [1.0262Ih 1.63' 0.20' 4.5w 0.464k 2.43'
surface tension, cloud mN/m point! "C 71
none none
71
none
62
none
62
0
O.l@
0.51
1293 k 18
1.03
1552 f 41 >0.40
2.06"
1331
1.29' 1.74m 3.28'
0.428
aData taken from refs 1-5 and 45-49 unless otherwise noted. *Refersto the substituents, &, Rz, and R3, on the basic glycopyranose units of the fkyclodextrin molecule as shown:
Average molar substitution is defined as the average number of hydroxyethyl, hydroxypropyl, or methyl groups present per glucopyranose unit. For example, the value of 0.51 for HE-6-CD means that there are on average 0.51 hydroxyethyl groups present per glucopyranose unit (or a total of 3.6 such groups per one cyclodextrin molecule). The molecular weight and degree of substitution of 8-CD derivatives can be determined via use of fast atom bombardment mass spectrometry (see refs 37,38,54, and 59). "Unlike the native, unmodified @CD,which is a single product, the modified @CDs typically contain numerous homologues and isomers of the products that are symmetrically distributed around an average molecular mass (see refs 37,38,46, and 59). eThesurface tension values were taken from refs 48 and 50 and are for 0.10 (w/v) % solutions of the indicated 6-CD-containing medium. fThe cloud point temperature refers to the temperature at which the P-CD-containing solution turns turbid upon heating due to crystallization. *Aqueous solubility of 8-CD determined in this work. See ref 51 for data on the solubility of 6-CD at different temperatures. hLiterature value taken from ref 52. This reference gives data on the dependence of viscosity upon 8-CD concentration. 'Solubility of 6-CD in 4.0 M urea solution. Viscosity value indicated is for this same solution. jSolubility of 8-CD in 8.5 M urea solution. Viscosity value indicated is for this same solution. kTaken from ref 53. 'Value obtained using a 0.20 molar solution of the indicated b-CD derivative. "'Value obtained using 0.39 M solution of HP-P-CD. "Value taken from ref 38. OCloud voint temverature varied from 48 to 93 "C deDendine uvon the DOM-B-CD concentration (see text and ref 45). analyte (fixed concentration, typically 5 X 10" or M) at ita wavelength of maximum emission as a function of different cycldextrin concentrations (ranged from 0.00 to 0.014 M in the case of native 8-CD or 0.00 to 0.20 M in the case of the solubilized or derivatized p-CD systems) and analyzing the data on the basis of a modified Benesi-Hildebrand equation for 1:l or 2 1 inclusion complexes (19, 41, 42). Alternatively, in the TLC method, equilibrium binding constants were determined by measuring the retardation factors (R) of the analyk on polyamide TLC sheets (Baker or Brinkmannj as a function of the cyclodextrin concentration in the mobile phase and analyzing the data using the Armstrong-Nome-Stine type equations for 1:l or 2:l complexes (43,44).The binding constants were determined from the slopes and intercepts of the appropriate plots of the TLC or fluorescence data obtained using at least six different cyclodextrin concentration levels.
RESULTS AND DISCUSSION Description of Different 8-Cyclodextrin Systems. Table I gives the structures as well as compares and contrasts some relevant physicochemical properties of the different soluble p-CD systems to that of the native p-CD. It is important to note that native p-CD contains 21 hydroxyl groups. Consequently, upon chemical modification, the products obtained are always a mixture of the derivatized @-CDwith various degrees of substitution (4,37,38,46,51-53,60).Such mixtures can be characterized by mass spectrometry (37,523, 54). The mass spectrum of the 2-hydropropyl-8-cyclodextrin preparation utilized in this work is shown in Figure 1 and shows that a relatively symmetric peak distribution occurs around an average molecular weight of the product (ca. 1552) having a degree of substitution (DS) of 7.2. This corresponds to an average molar substitution of 1.03,i.e. each of the seven glucose units which comprise the native ,%cyclodextrin molecule contains on average 1.03 (out of a total of three possible)
Ii.
s. 91.
B
M.
n.
n. 6.
u. SI.
B 45. 41.
IMO
,600
W E (D.lron>
Flgure 1. Mass spectrum of the commercial (P-hydroxypropyl)-pcyclodexbln material (with an average molar substkutlon value of 1.03) employed in this work.
2-hydroxypropyl moieties. Although one would assume from the commercial catalog information that heptakis(2,6-di-0methyl)-p-cyclodextrin is a pure preparation, a recent study has demonstrated that it also contains a mixture of homologues (38). Fortunately, by carefully controlling the derivatization reaction conditions, batch reproducibility of the product-derivatized 8-cyclodextrins obtained is possible (38). The average molecular weight and molar substitution of the modified p-CD derivatives utilized in this work as determined from reported mass spectral data are summarized in Table
I. As also seen in the table, the aqueous solubility can be greatly increased by solubilization of native p-CD in aqueous
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
2827
Table 11. Apparent Binding Constants for Complexes of Various Analytes with 8-CD and Its Derivatives" Kb, M-lb
analyte
j3-CD
j3-CD/4.5 M urea
diazepam digoxin dansylthreonine dansylvaline ethyl-4-biphenyl acetate p-aminobenzoic acid 4-aminophthalamide 1-naphthol 2-naphthol indole nystatin phenolphthalein C8H5CH(CHJCO2-p-N02C,Hb
200 f 30 [220]' [I1 OOO]' 120 f 15 [64Id [l80le 125 f 21 [58Id [20Ole [3000]' 290 f 25
68 f 14
500 f 60 21 f 20 [184]f
HE-b-CD [140]' [5600]'
107 f 24 163 f 30 [2400Ic 140 f 15 38 f 8 503 f 35 141 & 15 399 25 36 f 5
*
HP-j3-CD
DOM-b-CD
138 f 28 [170]' [7300]' 156 & 30 68 f 18 [4100]' 388 f 22 160 10 2150 f 60
[770]' [370001' [I2 5001'
*
114 f 12
[ 22 0001g [5881
175 f 15 328 f 20 [2350018 [3300Ih
Binding constants are for 1:l j3-CD-analyte complexes. Binding constants determined in this work. Literature values where available are given in brackets. These represent averaged Kb values for the interaction of the analyte with the derivatized j3-CD formulations as described in Table I. cValues taken from ref 54. dValues taken from ref 58. eValues taken from ref 19. fValue taken from ref 27. #Values taken from ref 59. Values taken from ref 56. urea solution (44) or via the use of the chemically modified P-CD derivatives (4, 45-50). Compared to its solubility in water alone (51), the solubility of native P-CD in water is 6-12-fold greater if urea is used as solubilizer while the 6-CD derivatives are more soluble by factors of 25 or greater. It is interesting to note that although the solubility of native j3-CD increases with temperature (4, 51), it decreases very for sharply for the methylated DOM-6-CD derivative (45,48), which the cloud point (i.e. critical temperature at which the clear homogeneous solution becomes turbid due to precipitation of the DOM-B-CD) ranges from 48 ([DOM-@-CD]= 0.20 M) to 93 OC ([DOM-0-CD]= 0.001 M). Thus,one should avoid elevated temperatures when this particular P-CD derivative is used in any luminescence applications. In contrast with native 0-CD, both HP-0-CD and DOMP-CD are surface active and cause a considerable decrease in the surface tension of water (54). The viscosity of aqueous native @-CDsolutions does not differ significantly from that of water (52). However, as seen from the data presented in Table I, the viscosity of urea-solubilized (3-CD solutions (44, 49) or solutions of the derivatized P-CD is considerably greater (50) and increases with concentration. In terms of volume handling/manipulation considerations, the HP-@-CDwould appear to have an advantage since at each concentration level, it has the lower solution viscosity compared to the other water-soluble @-CD-containingmedia. Lastly, it is important to note that the inclusion complexing ability exhibited by the native @-CDmolecule is, although modified, still largely retained in the different water-soluble P-CD systems (53-56). Table I1 summarizes some analyte@-CDbinding data for the inclusion complexation process which serves to illustrate the general complexing abilities of the different systems. In general, compared to that observed for native @-CDalone, the binding constants are reportedly decreased in the urea-solubilized 0-CD medium and the Kb values were found to decrease as the urea concentration was increased (44). This is presumably due to the fact that the urea competes with the analyte molecule for cavity binding sites. There are exceptions to this general rule however as seen from the dansylvaline and indole data in Table 11. Likewise, the HE-P-CD preparation exhibited a diminished complexing ability relative to b-CD which was postulated to be due to steric hindrance caused by the introduction of the hydroxyethyl moiety at the mouth of the CD cavity (54). In most cases, DOM-P-CD was found to bind analytes the strongest (54,56). This was attributed to its increased hydrophobic character (56). HP-P-CD reportedly has an intermediate analyte complexing tendency compared to that of
Table 111. Calculated Values of Amount of Analyte Complexed as a Function of 8-Cyclodextrin Concentration
[b-CD], M 0.005 0.010 0.100
0.200 0.400
% of analyte complexed" Kb = IO0 M-' Kb = IO M-'
33.3 50.0 90.9 95.2 97.6
4.76 9.09 50.0 66.7 80.0
a % analyte complexed calculated for 1:l inclusion complexes as shown in eq 1 for indicated Kb values assuming [analyte] = 1.00 X IO+ M. ~~
native 8-CD and HE-P-CD (54,37). As can be seen from the data in Table 11, HP-8-CD preparations generally bind analytes better than does HE-8-CD but slightly worse compared to native P-CD. However, for some analytes, HP-P-CD exhibits an equal or greater complexing ability than does native 8-CD (Table 11). It has been reported that the relative complexing ability of B-CD derivatives is affected by thier degree of substitution (i.e. average molar substitution value, Table I). Typically, the lower the degree of substitution, the better is their analyte-complexingability (55,57). Obviously, in the case of derivatized P-CDs, both the steric (which inhibits complexation) and the hydrophobic (which favors complexation) effects are in opposition with respect to the analyte inclusion complexation process and thus a range of binding tendencies are observed (54-56). As recently nicely summarized, for the substituted @-CDs,"the most noticeable difference is that the substituted CD can prevent a bulky analyte from completely entering the CD cavity, while offering better protection to smaller sized analytes" (37). It should also be noted that the analyte orientation within the CD cavity can be different for its interaction with derivatized 8-CDs compared to that observed in native 8-CD (60) and that higher order complexes are possible at the higher CD concentrations (43, 44). Despite the differences in complexing ability, all of the soluble fi-CD materials examined should be able to sufficiently complex most analytes such that at their higher obtainable concentrations (relative to that of native P-CD), a greater fraction of the analyte is complexed. As shown in Table 111, the amount of analyte complexed (for a Kb value of 10 M-') increases from 4.76 to 66.7% as the 8-CD concentration increases from 5 to 200 mM. Thus, at the higher CD concentrations, a greater fraction of the analyte is present within, and protected by, the CD cavity which in many cases should
2928
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
Table IV. Enhancement of Fluorescence of Dansyl Amino Acids in the Presence of Various Cyclodextrin Media"
0.014 M
analyte
fluorescence enhancement factorb 0.10 M 0.20M j3-CD in j3-CD in 0.20 M 4.0 M urea 8.5 M urea HE-B-CD
H,O
MeOH
8-CD
dansylthreonine
1.0
23.0
11.4
18.8
Ne-dansyllysine dansylglycyl-L-tryptophan dansylsarcosine monodansylcadaverine dansyl-D,L-norleucine dansylglycine didansyllysine N,N-didansyl-L-cystine didansyl-l,4-diaminobutane
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
16.5 20.5 21.8 18.1 14.0 11.5 11.4 18.0 15.3
8.1 7.9 24.2 8.2 7.8 6.9 4.9 9.6 6.5
12.5 13.0 34.7 13.2 11.4 10.0 8.4 14.9 10.9
didansylhistidine tridansylspermidine dansylestradiolf dansyllysozymeB
1.0 1.0 1.0 1.0
3.5 0.9
2.2 0.9 5.6 2.1
3.3 0.5
15.5
3.4
16.7 17.6
12.9 17.5
10.4
8.5
0.20 M 0.20 M HP-@-CD DOM-8-CD 19.2 [25.4Ic 16.7 20.0 19.7 14.5 13.6d 10.6 9.9 11.9 14.5 [13.7]' 3.3 0.8 8.2 4.4
16.2 18.0
13.3
'@-Cyclodextrin media examined are summarized in Table I. *Enhancementfactor in indicated medium relative to that observed for the same analyte under same experimental conditions in water alone. The value is the average obtained at at least two different analyte concentration levels. Measurements were made using A,, = 365 nm and A,, 510-525 nm depending upon the medium. cValue obtained in a 45% of HP-0-CD. dValue obtained in a 4.5% HP-j3-CD solution. 'Value obtained in a 4.0% HP-8-CD solution. 'Dansyl derivative prepared using modified procedure as described in ref 39. BDansyl derivative prepared using a modification of procedure described in ref 19. result in a greater fluorescence output. Fluorescence in Different 8-CD Media. Addition of the different P-CD media to aqueous solutions of the various test analytes typically resulted in significant shifts in the emission wavelength maximum and enhanced the fluorescence intensity observed compared to that in bulk aqueous solution alone. Figure 2 shows these trends for N-phenyl-1-naphthylamine as the analyte. As can be seen, the emission wavelength was significantly blue-shifted (from 465 nm in water to 408 nm in DOM-P-CD) and the fluorescence intensity enhanced in the P-CD media (or bulk methanol). Although not shown, as the concentration of P-CD (or derivatized 0-CDs) was increased, the fluorescence intensity of the naphthylamine progressively increased while the emission peak shifted more to the blue. Similar results were observed for many of the other analytes examined. For example, the emission wavelength of dansylamino acids was blue-shifted from ca. 540-559 nm in water to 512-525 nm in the different 8-CD media with fluorescence intensity enhancements in the range 2-24 (Table IV). Likewise, the emission wavelength maxima for the analytes indole, 2-methylhydronaphthoquinone, methyl salicylate, procaine, and quinizarin shifted from 348,430,360, 355,and 567 nm, respectively, in water to 339,420,354,340, and 554 nm, respectively, in the presence of the 6-CD media. Concomitant with these blue shifts in emission wavelength in the presence of the 0-CDs were enhancements in the fluorescence intensity (see Table V). Although addition of 8-CD, urea-solubilized 8-CD, and the different soluble 8-CD derivatives to dansylamino acids (Table IV) or aromatic compounds/pharmaceuticals (Table V) typically always resulted in enhanced fluorescence compared to that observed in bulk water alone, the relative magnitude of this enhancement was found to be dependent upon the specific 8-CD system employed. This is presumably due to their different complexing tendencies as previously mentioned. For the dansylamino acids (Table IV), it is seen that the presence of 0.014 M P-CD enhanced the fluorescence output by factors ranging from 2.2 to 24.2. These P-CD enhancement results are in good agreement with literature reports (19-24). As previously noted by Kinoshita (211, the use of higher 8-CD concentrations (i.e. urea solubilized P-CD) can still further intensify the emission observed from dansylamino acids. As
4.0
,3.0 .U r(
E
L1 W
d
P8 W
k
2 $ 2.0 3 U
9
3
2
1.0
Fluorescence emission spectra of 3.55 X lo-' M N phenyl-1-naphthylamine with an excitation wavelength of 340 nm in different media: (1) water; (2)0.014 M PCD; (3)0.20 M PCDI8.0 M urea; (4) methanol; (5)10% (w/v) HP-P-CD; (6)0.20 M DOM-P-CD. Figure 2.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991 Table
2929
V. Fluorescence Enhancement Data for Organic/Pharmaceutical Solutes in Different 8-Cyclodextrin Media fluorescence enhancement factorb 0.10 M 0.20 ~.M . -..~ 0.014 M 8-CD in 8-CD in 0.20 M 0.20 M B-CD 4.0 M urea 8.5 M urea HE-B-CD HP-8-CD ~~
analyte
conditionso
H20
MeOH 1.50
salicyclic acid methylsalicylate
(302/404), pH 9 (303/356)
1.00 1.00
4-aminophthalimide
(265/525), DMSOd
1.00
12.7
1.35 1.60 [1.78Ic 11.9
4-amino-N-mehylphthal- (265/525), DMSOd imide N-methylaniline (280/350), MeOHd
1.00
N-phenyl-l-naphthylamine p-aminobenzoic acid
(340/444), MeOHd
1.00
(305/334), 10 mM NaOHd procaine (292/340), MeOHd 4-(N,N-dimethylamino)- (295/360), MeOHd benzonitrile
1.00
fenproporexj indole
(260/560), pH 7 (277/340), MeOHd
1.00 1.00
DAPI' 2-methylhydronaphthoquinone
(320/460), pH 3 (312/427), pH 3.5
1.00 1.00"
quinizarinp
(470/580), 90% 1,4-dioxane/pH 7.1d
1.00
4.20
[2.60]" 3.60
1,8-dihydroxyanthraquinone prazepam diazepam 1-naphthol 2-naphthol pamoic acid' 5-methoxypsoralen aflatoxin B, chlortetracycline bombesin furosemide
(435/570), dioxaned
1.00
1.70
[3.3]4 1.80
3.40 1.00 1.00 3.20 1.00 200* 15 1.00 1.00 1.00 13.5 1.00 3.40 1.00 2.00 1.00 2.00 1.00 0.63
ribonuclease diphenyl phosphate retinol
(360/500), MeOHd (360/500), MeOHd (282/350), MeOHd (310/350), pH 6.4 (240/510), pH 11 (317/490), MeOHd (365/418), MeOHd (345/530), pH 8 (280/340), pH 7 (350/400), 90% MeOH/ 10% HZ0 (pH 7)d (275/310), pH 6 (254/290), pH 7 (366/530), MeOHd
retinal a-tocopherol nystatin merocyanine 540 erythrosin
(381/433), MeOHd (299/328), DMSOd (306/400), MeOHd (474/573), MeOHd (533/548), MeOHd
V
17.5
11.5
15.9
3.0 [3.l]g 6.3
1.00
1.00
1.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00
2.49 14.7 3.8
1.36 [3.0Ih 7.9 [9.9] 4.2
[3.5]' 1.9 1.40 [1.4Ik 26.5 [la]"' 1.65 2.88 0.9 1.04
6.1 1.3 2.10 [ 1.501"
1.50 1.00 27.0 1.70 1.28 7.60 [8]I 35.3 1.5 1.15 1.25 3.38 2.8 [3.0]' 1.1
1.34 9.8
1.23 8.4 [we 10.9
7.2
8.5
1.64
1.64
18.1
2.5
5.00
17.0
0.39 2.40 1.80 1.10
0.50 3.30 2.40
4.90
3.5
3.8
18.5
31.4
[8.5]f
22.9
0.24
1.3 1.1 13.7 3.72
10.50
3.10 3.80
4.00
4.20
1.05
1.90
1.60
3.70 2.10 20.9
2.50 1.20 10.2 2.10
5.70 2.30 43.6
3.10
1.82 8.3 35.6 2.4 1.47
2.90 1.15 33.2
9.0 48.3 2.3 0.70
0.33
3.83 9.0 47.2 0.5
3.8 1.7 1.4
2.60 4.9
0.40 4.0
1.10 3.60 2.90 1.60
[O. 171 1.10 7.10 3.20 1.70
2.00 4.10 4.20 0.60
[I.O]W
6.10 13.2 2.00 5.70
15.5
5.6
19.7
4.40
1.6 1.35 1.60
2.60
[19.411
10.7
3.10
19.1
11.9
0.06
2.28
1.8 1.10
3.40
3.30
1.47 1.81
1.26
1.18
2.40
0.75
0.20 M DOB-8-CD
3.70 1.60
aAny special experimental conditions are noted. The numbers given in parenthesis refer to the excitation and emission wavelengths, respectively, for the indicated analyte. *Enhancement factor in indicated medium relative to that observed in water alone under otherwise identical experimental conditions. The reported value is the average of duplicate determinations at two different analyte concentration levels. Value estimated from literature data (62). dIndicates the solvent used for preparation of concentrated stock solution of the analyte. eValue obtained for 0.15 M j3-CD in 8.5 M urea. /Value obtained in 4.5% (w/v) solution of HP-&CD. #Value taken from literature (63). hLiterature value (64). 'Literature value (17). j (*)-3-(a-Methylphenethylamino)propionitrile. Value estimated from literature (27). I4',6-Diamidino-2-phenylindole dihydrochloride. Value taken from the literature (65). "Value obtained using 7030 (w/v) water:methanol as bulk solvent. OValue estimated from literature data (40). P1,4-Dihydroxyanthraquinone. q Value estimated from literature data (28).'4,4'-Methylenebis(3-hydroxy-2-naphthalenecarboxylic acid). 'Value taken from the literature (30). 'Value estimated from literature data (66). "Value taken from the literature (67). "No fluorescence signal detected in water. Relative fluorescence signals, taken from the literature (67).
seen from the data in Table IV, the fluorescence in the presence of 0.10 M P-CD/4.5 M urea is ca. 1.5 times that observed in 0.014 M P-CD. Although the data are limited, similar enhancement results were observed using 0.20 M HE-P-CD. The use of 0.20 M HP-P-CD resulted in an approximately 1.7-fold enhancement for these dansylamino acids compared to that in native P-CD. Results comparable to that for HP-P-CD were obtained using 0.20 M @-CDin 8.5 M urea or 0.20 M DOM-P-CD. The use of any of the different water-soluble 8-CD media in which the CD concentration is 0.20 M allows one to approach fluorescence outputs typically observed only in a pure organic solvent such as methanol (Table IV). However, of these different concentrated P-CD
systems, the HP-(3-CD is more convenient in terms of volume handling/manipulation considerations since its solutions are less viscous compared to the others (Table I). The use of the more concentrated HP-@-CDmedium in lieu of native @-CD in fluorometric assays for dansylated amino acids leads to better sensitivity which is comparable to that previously reported for urea-solubilized @-CD(19). Also, compared to results using j3-CD alone (61), enhanced luminescence and better detection limits for the dansylamines were achieved when the more concentrated HP-@-CDmedium was used as a spray reagent for fluorometric detection on developed thin-layer chromatographic plates. Additionally, as shown in Table V, fluorescence enhance-
2930
ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991 2.0
t 1.01
0.5
i
/
/
J
/
1.5
6
P Figure 3. Relative fluorescence intensities of 3.4 X lo-' M didansyllysine (Aex = 365 nm, X, = 515 m:sdM lines)and 7.1 X 10" M 4-amin~N-methylpllmide (X, = 265 nm, X, = 525 nm; dashed lines) in the presence of varied concentrations of 6-cyclodextrin ( 0 ) or (hydroxypropyl)-P-cyclodextrin (A).
ments in 6-CD media were observed for a variety of other aromatic compounds. The enhancement factors ranged from 1.1to 48.3. For all but four of the 33 compounds examined, the use of the different soluble 6-CD media at concentrations of 0.045 M or greater resulted in maximum enhancements. Of the different soluble 0-CD systems, the use of HP-0-CD elicited the greatest emission output from the 13 compounds, that of DOM-P-CD, 11, and that of urea/p-CD, 4. The magnitude of the enhancement observed in all of these media was found to depend upon the CD concentration. As shown in Figure 3 for didansyllysine and 4-amino-N-methylphthalimide, the typical fluorescence intensity-native p-CD concentration behavior observed was an increase in the intensity with concentration until the P-CD water solubility limit was reached (left-hand portion of Figure 3). In the case of HP-8-CD (right-hand part of Figure 3), the emission intensity of didansyllysine increased with increasing concentration until it leveled off a t a maximum plateau value whereas for 4amino-N-methylphthalimide, a maximum in intensity is observed a t an optimum HP-0-CD concentration. Behavior similar to that just noted for HP-0-CD was observed when the urea/p-CD, HE-0-CD, or DOM-0-CD media was used. For the urealp-CD system, it was noted that increasing the urea concentration at a fixed p-CD concentration resulted in a gradual diminution of the intensity for some analytes, presumably due to "quenching" by the urea, as had previously been reported for dansylamino acids (21). This is probably a consequence of the fact that the urea competes with analyte molecules for binding sites in the protective cyclodextrin cavity. The fact that many cyclodextrin-analyte binding constants reportedly decrease as the urea concentration is increased (44) lends support to this possibility for the observed diminution in fluorescence intensity. As illustrated for the polyene antibiotic, nystatin, in Figure 4,the enhanced fluorescence emission observed in the presence of the different CD media leads to corresponding increases in the slopes of the intensity-analyte concentration calibration graphs, which allows for more accurate determination of lower analyte concentrations. For example, the detection limit for nystatin is a factor of 4 lower if the assay is conducted in HP-/3-CD compared to that possible in native P-CD alone. I t is important to emphasize that although four analytes (fenproporex, indole, diphenylphosphate, and retinal) exhibited their greatest emission intensity when in the presence of 0.014 M p-CD, this does not mean that p-CD is necessarily the best medium to use in fluorometric assays for those compounds or that the enhancement factors listed in Table V for (3-CD are actually obtainable when real samples are
E
w 1.0
[ 4
%
s
0.5
107[NYSTATIN], M
Figure 4. Standard calibration curves of relative fluorescence intensity
vs analyte concentration for nystatin in different media: (1) water (0); (2)0.0142M P-cyclodextrln (0); (3)0.10M /ikyclodextrin in 4.1 M uea (0); (4) 10.0% (w/v) (2-hydroxypropyl)-~-cyclodextrin (V). The emission and excitation wavelengths were 400 and 306 nm, respectively.
analyzed. The reason for this stems from the fact that unless one is willing to take the time (from 15 to over 60 min dissolution time is required in order to achieve an equilibrated >0.016 M 0-CD solution depending upon the source of the CD, the temperature, and whether the hydrate or anhydrous form is employed (51))and trouble to add solid 0-CD to real aqueous sample solutions, it will be necessary to add an aliquot of a concentrated p-CD solution to the analyte solution in order to have an adequate CD concentration necessary for enhanced fluorescence. However, this addition will also serve to dilute the analyte solution. The fact that an aqueous saturated p-CD solution is only 0.014 M 8-CD means that if one made a 1:l dilution, the final p-CD concentration would only be 7 mM (which would mean that lower enhancement factors compared to those presented in the tables for 0.014 M p-CD would result) and the analyte concentration would have been halved. Thus, it would take a P-CD enhancement factor of 2 just to break even and achieve the same fluorescence intensity as in water alone. However, the ability to use the more concentrated P-CD-containing stock solutions (Le. 0.20-0.40 M) which can be prepared from the P-CD derivatives or urea-solubilized p-CD system virtually eliminates these dilution effect problems. Their general use in lieu of native p-CD should thus be considered in most fluorometric assays. The only exception to this general guideline would be those instances in which P-CD spray reagents are used for TLC detection purposes (23,61) since no dilution of the analyte occurs in such an application.
ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991 2931
2.0
c
40
t -
~~
20
40
60
T h , min.
I
0
1
4
I
8
12 16 20 7. ("1") Acetaritrile
,
24
I
28
Figure 5. Relative fluorescence intensity (Ae, 338 nm, A, 521 nm) of 3.99 X lo-' M dansyiglycine as a function of acetonitrile concentration ( % (v/v)) in the absence of any added cyckdextrin (lower curve (D)),and in the presence of 0.01 M j3-CD (middle curve, (O)), and 0.08 M HP-P-CD (top curve (A)).
The use of the different water-soluble j3-CD systems is also superior to that of p-CD alone for most applications involving postcolumn addition of the CD to the chromatographic mobile phase in order of enhance HPLC fluorescence detection. Typically, in reversed-phase HPLC separations, methanol (or acetonitrile)-water hydroorganic mobile phases are employed. In addition to the dilution effect just mentioned, the presence of either methanol or acetonitrile in the mobile phase leads to another complication with respect to the degree of enhanced fluorescence obtainable with 8-CD, since the organic solvent present will compete with the analyte molecule for j34D cavity binding sites (27,30,58,68,69) which reduces the percentage of complexed (and protected) fluorophore. As shown by the middle curve in Figure 5, the fluorescence intensity of dansylglycine becomes progressively weaker as the percent of acetonitrile in an aqueous 10 mM 8-CD solution increases. In fact, at acetonitrile concentrations greater than ca. 30%, the fluorescence intensity observed in the aqueous acetonitrile medium becomes equivalent to that observed for the same solution containing 10 mM P-CD. If 2.5 mM j3-CD is employed, the two curves become equal at about 24% acetonitrile (58). Under those conditions, no benefit would be accrued by the addition of (3-CD with respect to fluorescence detection. In contrast, as shown in the uppermost curve in Figure 5, the use of 80 mM HP-P-CD would still yield a fluorescence enhancement factor of 2 in the presence of 28% acetonitrile. In fact, the two curves do not become equivalent until the ace-
6. ~lu~rescence vs t h e stabwy proms of the lysine dedvative with o-phthaldialdehyde/2-mercaptoethanol in different media: (A) water: (B) 0.014 M j3-CD (C) 0.20 M HP-BCD; (D) 0.20 M DOWj3-W. Conditions: pH = 9.0, A,, = 350 nm, A, = 445 nm.
tonitrile concentration reaches 45%. The use of a still higher HP-j3-CD concentration level would allow one to further extend the acetonitrile range over which enhanced fluorescence is still observed. Results similar to those mentioned for HP-j3-CD were observed if 0.10 M DOM-8-CD was used in such an experiment. For postcolumn detection applications, the use of these latter two j3-CD derivatives instead of j3-CD allows for greater fluorescence enhancements a t higher percentages of the organic component in the chromatographic hydroorganic mobile phase. Of these two derivatives, HP-j3CD is more convenient to employ in terms of volumetric handling due to its lower solution viscosity. Lastly, it is important to note that in addition to enhanced fluorescence, the use of HP-j3-CD or DOM-8-CD can in some instances enhance the stability observed for a particular luminescence system. For example, Roth's method for the determination of primary amino compounds is based upon the reaction of the amino compound with o-phthaldialdehyde (OPA) in the presence of 2-mercaptoethanol (ME) which yields fluorescent isoindole products. A problem with this assay stems from the fact that many of the isoindole products formed are unstable and exhibit a significant decrease in their fluorescence intensity with time (70). Preliminary results presented in Figure 6 for the lysine OPA/ME derivative show that in addition to enhancing the relative fluorescence output, the use of HP-@-CDappears to yield a much more stable fluorescent system with the signal being fairly constant for up to 1 h after initiation of the derivatization reaction. In the case of DOM-@-CD, the fluorescence signal becomes constant after about 45 min. Similar results were obtained
2932
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
Table VI. Fluorescent Data for Selected Analytes in Different Media medium HZO D2O
50:50 H20:ethanol methanol ethanol acetonitrile [or THF] aq 0.014 M 8-CD aq 0.20 M HP-8-CD
relative fluorescence intensity of indicated analyte’ 1,5-DNSAd retinol nystatin procaine
DNS-tryb
4-AMPIC
1.0 2.5
1.0
1.0
5.3
1.9
5.5 7.4
17.5 20.4
1.0
1.0
1.0
7.0 7.9 7.9
2.1
13.2 17.1
14.7
[23.9]
20.1
4.8
1.1 4.9
2.4
7.9 8.5
DAPI‘ 1.0 1.4
5.18 12.3
11.5 15.5
7.1
1.0
4.5
8.3 2.8
coumarin If
26.5
1.7
13.7 18.7 1.4
13.7
a Relative fluorescence intensity in indicated medium compared to that observed in water alone under the same experimental conditions. Refers to dansyltryptophan. Refers to 4-amino-N-methylphthalamide. Refers to l-(dimethylamino)-5-naphthalenesulfonamide.e Refers to 4’,6-diamidino-2-phenylindole. ’Refers to 7-(diethylamino)-4-methylcoumarin; measurements made at A,, = 372 nm, ,A, = 451 nm. gValue obtained at a [P-CD] = 0.01 M.
with ornithine as the analyte. Further work is obviously required to determine the scope of this particular application. In addition, the stability of the dansyl chloride labeling reagent (for amino compounds) was found to be further increased in the presence of the more soluble @-CDderivatives compared to that in native @-CDalone, as had been previously noted for urea-solubilized P-CD in the literature (20, 23). Although more work is required, these preliminary results indicate that use of these more concentrated P-CD media might prove to be very effective in increasing the stability of fluorescent reagents required in and/or products formed in many liminescent derivatization reactions. Mechanism(s) for t h e Enhanced Fluorescence. Fluorescence enhancements observed for the different analyte molecules upon inclusion complexation with CDs has been previously ascribed to an increase in the radiative rate constants (63), decrease in degrees of freedom in molecular motion (4, 13, 63, 72), prevention of collisional deactivation (64), restricted conformations (62), favorable microenvironment polarity effects (4, 13,63, 71, 72), and shielding of the excited state from water molecules (63, 71) or other species present in the buik aqueous solution (72), among others. In particular, excited states of analyte molecules possessing a -OH or -NH moiety are efficiently quenched by water molecules due to formation of exciplexes from the interaction of the excited state of the fluorophore with water molecules (63,71). Thus, the fluorescence enhancements observed for many of the analytes in this work (Le. the dansyl amino acids, indoles, procaine, N-methylaniline, etc.) are probably principally due to elimination of or a reduction in their exposure to water molecules upon complexation with the added P-CD media (31, 63, 71). As seen from the data in Table VI, there is an increase in the relative fluorescence intensity observed for the indicated analytes as the hydrogen-bonding ability of the bulk solvent decreases. Other mechanisms are operative for other types of analyte molecules. For instance, in water alone, diphenyl phosphate’s conformation is such that it exists as an intramolecular excimer. Upon complexation with P-CD materials, excimer formation is prevented and the more intense monomeric fluorescence band observed (66). Similarly, the use of @-CD media to control an analyte’s aggregational equilibria can often result in enhanced fluorescence. This is due to selective P-CD complexation of the monomeric form of the analyte which is typically more highly fluorescent. The analytes 1,8-dihydroxyanthraquinone and erythrosin exhibit monomeldimer equilibria in aqueous solution while retinol forms micellar aggregates (67, 73, 74). These equilibria are all shifted toward the monomer side of the equilibrium upon complexation with P-CD, and as a consequence, all exhibit intensified fluorescence. In addition, the increased solution viscosity exhibited by the urea/p-CD system or derivatized P-CDs (Table I) could
account for some portion of the fluorescence enhancements observed for some analytes. Of course, in many instances, a combination of factors will be operative to give rise to the @-CD enhanced fluorescence observed for a particular analyte molecule. As has been pointed out by a referee, for the @-CDlurea system, the enhancement observed for some analytes may be due to the presence of the urea, which could serve to displace some of the quencher water molecules from around a hydrophobic analyte molecule, thereby enhancing the analyte’s fluorescence intensity, as has been recently discussed in the literature (75). Indeed, a brief study revealed that the presence of urea (in the absence of any CD) enhanced the fluorescence of some analytes relative to that observed in water alone. For example, the fluorescence of 1-naphthol was enhanced by a factor of 12.0 in the presence of 4.0 M urea, with this enhancement factor being further increased to 19.1 in the 0.10 M 6-CDI4.0 M urea system. This clearly indicates that for some analytes, both the presence of the solubilizing agent, urea, and the solubilized @-CDcontribute to the intensified fluorescence observed. ACKNOWLEDGMENT
We thank Hong Wang (Wake Forest University) for determining some of the binding constants presented and Rick Strattan and Steve Herschleb (both of Pharmatec, Inc.) for helpful discussions. A reviewer is thanked for pointing out ref 75. Registry NO.P-CD, 758539-9;DOM-P-CD, 51166-71-3;DAPI, 47165-04-8; dansylthreonine, 35021-16-0;N,-dansyllysine, 110184-4; dansylglycyl-L-tryptophan,19461-22-4; dansylsarcosine, 1093-96-5; monodansylcadaverine, 10121-91-2; dansyl-DL-norleucine, 61417-01-4; dansylglycine, 1091-85-6; didansyllysine, 1263-03-2;N,N-didansyl-L-cystine, 18468-46-7;N,N’-didansyl1,4-diaminobutane,13285-10-4;N,l-didansylhistidine, 1110-87-8; tridansylspermidine, 66039-59-6; dansylestradiol, 30808-48-1; lysozyme, 9001-63-2; salicylic acid, 69-72-7; methyl salicylate, 119-36-8; 4-aminophthalimide, 3676-85-5; 4-amino-N-methylphthalimide, 2307-00-8; N-methylaniline, 100-61-8;N-phenyl-1naphthylamine, 90-30-2;p-aminobenzoic acid, 150-13-0;procaine, 59-46-1; 4-(N,N-dimethylamino)benzonitrile, 1197-19-9; fenproporex, 15686-61-0; indole, 120-72-9; 2-methylhydronaphthoquinone, 481-85-6; quinizarin, 81-64-1; l&dihydroxyanthraquinone, 117-10-2; prazepam, 2955-38-6; diazepam, 439-14-5; 1-naphthol, 90-15-3; 2-naphthol, 135-19-3;pamoic acid, 130-85-8; 5-methoxysporalen, 484-20-8; aflatoxin B1,1162-65-8; chlortetracycline, 57-62-5; bombesin, 31362-50-2; furosemide, 54-31-9; ribonuclease, 9001-99-4;diphenyl phosphate, 838-85-7;retinol, 68-26-8; retinal, 116-31-4;a-tocopherol, 59-02-9; mystatin, 140061-9; merocyanine 540,62796-23-0;erythrosin, 16423-68-0; dansylvaline, 1098-50-6; dansyltryptophan, 19461-29-1; 1-(dimethylamino)-5-naphthalenesulfoxamide, 1431-39-6; 4,6-diamidino-2-phenylindole, 75980-76-6; 7-(diethylamino)-4methylcoumarin, 91-44-1.
ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991
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RECEIVED for review May 21,1991. Accepted September 6, 1991. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, and to Pharmatec, Inc. (Alachua, FL) for support of this research project. This work was presented at the 1990 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New York, NY, March 7, 1990 [Abstract No. 8111 and the 87th Meeting of the North Carolina Academy of Science, High Point, NC, March 31, 1990.
