Enhancement of Fluorescence in Thin-Layer Chromatography

Anal. Chem. 2000, 72, 1759-1766

Enhancement of Fluorescence in Thin-Layer Chromatography Induced by the Interaction between n-Alkanes and an Organic Cation Fernando P. Cossı´o* and Ana Arrieta

Kimika Fakultatea, Euskal Herriko Unibertsitatea, P.K. 1072, 20080 San Sebastia´ n-Donostia, Spain Vicente L. Cebolla,* Luis Membrado, Marı´a P. Domingo, and Patrick Henrion†

Departamento de Procesos Quı´micos, Instituto de Carboquı´mica, CSIC. Marı´a de Luna, 12. 50015 Zaragoza, Spain Jesu´s Vela

Grupo de Espectroscopı´a Analı´tica y Sensores, Departamento de Quı´mica Analı´tica, Facultad de Veterinaria, Universidad de Zaragoza, Avenida Miguel Servet 177, 50013 Zaragoza, Spain

Fluorescence enhancement of a broad variety of solutes has been used extensively in TLC although no thorough explanation has been proposed. In this work, we try to understand it and explore new applications to which it can be put. In this way, alkanes can be quantitatively determined by fluorescence scanning densitometry using silica gel plates impregnated with berberine sulfate. Molecular simulation and analysis of molecular orbitals allows this phenomenon to be explained in this case and lays the groundwork to explain fluorescence enhancements produced by other molecules. A ion-molecule interaction between alkanes and berberine sulfate is responsible for the enhancement of fluorescence produced by alkanes. Computational results suggest that the surrounding alkane molecules provide an apolar environment to the berberine cation, thus enhancing the intensity of the fluorescence signal. This proposed explanation has been tested by extending the fluorescence determination to other compounds. These include biologically interesting saturated and unsaturated fatty acids, steroids and derivatives, prostaglandins, ceramides, galactocerebrosides, as well as terpenes, and polypropylene glycols. In addition, according to the proposed explanation, the properties required for alternative impregnants to berberine are discussed. It has been long reported that the luminiscence of a broad variety of solutes adsorbed on TLC plates can sometimes be greatly enhanced by spraying or dipping the plates with a variety of organic solvents.1-3 From this, phosphorescence and fluores* Corresponding authors: [email protected]; [email protected]. † On leave from Laboratoire de Thermodynamique et d’Analyse Chimique, Faculte´ des Sciences, Universite´ de Metz, France. (1) Baeyens, W. R. G. In Molecular Luminescence Spectroscopy Methods and Applications: Part 1; Schulman, S. G., Ed.; Chemical Analysis Vol. 77; WileyInterscience: New York, 1985; Chapter 2. (2) Fenske, M. Chromatographia 1995, 41, 175-177. 10.1021/ac991302q CCC: $19.00 Published on Web 03/10/2000

© 2000 American Chemical Society

cence enhancements caused by paraffins have merited considerable interest.4,5 As an example, n-hexane increases TLC phosphorescence in the detection of certain drugs.4,6,7 In some cases, higher enhancements have been obtained using other solvents such as alcohols.4,5 With respect to fluorescence in TLC, enhancements with alkanes of the respective fluorescence responses of some alkaloids, polycyclic aromatic hydrocarbons (PAHs), and dansyl derivatives are reported to have been carried out on silica gel, alumina, and charge-transfer plates.5-10 In general, they involve dipping the dry, developed plate into a solution of dodecane in hexane, liquid paraffin in hexane, or liquid paraffin in ether. Likewise, it has been reported that the fluorescence response in TLC for identical amounts of a PAH is significantly greater on a octadecyl-bonded plate than for a silica gel plate.11 As an alternative to the use of saturated compounds as detection reagents, the phenomenon of fluorescence enhancement has also been used for the detection of alkanes, using berberine salts as a reagent.12-14 TLC plates can either be sprayed or dipped after development or impregnated before development since berberine salts are not eluted by most of the usual solvents in TLC. (3) Kartnig, T.; Goebel, I. J. Chromatogr., A 1996, 740, 99-107. (4) Miller, J. M.; Phillips, D. L.; Thornburn Burns, D.; Bridges J. W. Anal. Chem. 1978, 50, 613-616. (5) Uchiyama, S.; Uchiyama, M. J. Chromatogr. 1978, 153, 135-142. (6) Frijns, J. M. G. J. Pharm. Weekbl. 1971, 106, 865. (7) Bos, C. J. G. A.; Frijns, J. M. G. J. Pharm. Weekbl. 1972, 107, 111. (8) Alak, A.; Heilweil, E.; Hinze, W.L.; Oh, H.; Amstrong, D. W. J. Liq. Chromatogr. 1984, 7, 1273. (9) Funk, W.; Glu ¨ k, V.; Schuch, B.; Donnevert, G. J. Planar Chromatogr.-Mod. TLC 1989, 2, 28-33. (10) Ho, S. S. J.; Butler, H. T.; Poole C. F. J. Chromatogr. 1983, 281, 330. (11) Poole, C. F. In Quantitative Analysis Using Chromatographic Techniques; Katz, E., Ed.; John Wiley & Sons: New York, 1987; Chapter 6. (12) Mamlok, L. J. Chromatogr. Sci. 1981, 19, 53. (13) Huc, A. Y.; Roucache´, J. G. Anal. Chem. 1981, 53, 914-916. (14) Marsh, C. M.; Hiekane, C. J. J. Planar Chromatogr.-Mod. TLC 1991, 4, 293-298.

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It has been reported that the observed luminiscence enhancement depends, in general, on a number of factors, of which the most important are the sample studied, the characteristics of the sorbent layer, the enhancement reagent used and its concentration, and the time between impregnating the plate with reagent and making measurements. The possibility of interactions between sorbent and sorbate or between sorbent and the enhancing reagent has been mentioned.11 However, it has been pointed out that the phenomenon is not exclusive to TLC but also occurs in solution.5 Despite the observation of these phenomena, an explanation of the molecular mechanisms involved in this kind of fluorescence enhancement had never been proposed. According to previous works, enhancement is not related to the transmissibility of light through the thin layer, but rather to the properties of the reagent.5,15 According to this, alkanes would have adequate polarities, viscosities, and acidities to produce fluorescence enhancement. However, a more complete, molecular interpretation of these phenomena would be desirable in order to develop new interacting systems and take advantage of their analytical possibilities. Within this context, computational chemistry applied to theoretical chemistry may be a useful tool for explaining analytical phenomena and predicting new analytical systems. Thus, the aim of this work is to propose an explanation for the fluorescence enhancement in TLC produced by alkanes. This explanation has also served in our case for predicting the analyses of compounds other than alkanes and to propose other reagents for fluorescence enhancement. The approach used in this work combines computational and theoretical chemistry with experimental analytical studies, thus exploiting the synergistic interaction between both methodologies. EXPERIMENTAL SECTION Computational Studies. Self-consistent field (SCF-MO) calculations were performed using the PM3 method16 at the restricted Hartree-Fock (HF) level. The structures under study were fully optimized without symmetry constraints using the BroydenFletcher-Goldfarb-Shanno17 (BFGS) and Bartels18 algorithms. All stationary points were refined by minimization at the gradient norm of the energy at least below 0.1 kcal Å-1 deg-1 and characterized by harmonic vibrational analysis.19 All calculations were performed with the more precise SCF convergence and minimization criteria, according to the recommendations of Boyd, Stewart et al.20 Excited states were calculated by means of the configuration interaction-half-electron (CI-HE) method.21 Computer plots of the canonical frontier molecular orbitals reported (15) Uchiyama, S.; Uchiyama, M. J. Liq. Chromatogr. 1980, 3, 681-691. (16) (a) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 209. (b) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 221. (17) (a) Broyden, C. G. J. Inst. Math. Its Appl. 1970, 6, 222. (b) Fletcher, R. Comput. J. 1970, 13, 317. (c) Goldfarb, D. Math. Comput. 1970, 24, 23. (d) Shanno, D. F. Math. Comput. 1970, 24, 647. (18) Bartels, R. H. Report CNA-44 Center for Numerical Analysis, University of Texas, Austin, TX, 1972. (19) McIver, J. W.; Komornicki, A. K. J. Am. Chem. Soc. 1972, 94, 2625. (20) Boyd, D. B.; Smith, D. W.; Stewart, J. J. P.; Wimmer, E. J. Comput. Chem. 1988, 9, 387. (21) (a) Dewar, M. J. S.; Trinajstic, N. J. Chem. Soc. A 1971, 1220. (b) Dewar, M. J. S.; Olivella, S.; Stewart, J. J. P. J. Am. Chem. Soc. 1986, 108, 5771. (c) Dewar, M. J. S.; Jie, C. J. Am. Chem. Soc. 1987, 109, 5893.

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Figure 1. Fluorescent chromatographic response (area counts) with excitation wavelength for n-C16 (20 µg) on a berberine-coated silica gel plate (6 mg/100 mL of methanol).

in this work were performed with the SPARTAN program22 running on a Silicon Graphics O2 workstation. Molecular mechanics (MM) calculations were carried out with the AMBER force field23 as implemented in the MacroModel package.24 The conformer distribution of the different structures was explored by means of Monte Carlo simulations25 with a sampling of 1000 possible conformations. These calculations were performed on IBM RS6000 workstations. Samples and Standards. n-Alkanes (Fluka, Basel, Switzerland; Ultrascientific, North Kingstown, RI) were used as standards: octane (n-C8, 99+%); dodecane (n-C12, 99+%); hexadecane (n-C16, 99+%); octadecane (n-C18, 98+%); tetracosane (n-C24, 99%); triacontane (n-C30, 99+%); tetracontane (n-C40, 96%). The following compounds were also used: 5,R-cholestane (98.5%); 5,β-cholestan3R-ol (95%); cholesterol (99++%); cholesteryl stearate (99%); oleic acid (99%); a non-hydroxy-fatty acid ceramide (99%), from SigmaAldrich (Madrid, Spain). Geraniol (96%), stearic acid (97+%), and prostaglandin VII, (-)-3-hydroxy-6β-(3S-hydroxy-oct-1-enyl)-7R-pphenylbenzyloxy-2-oxabicyclo[3.3.0]octane, (96%) were supplied by Across Chimica (Geel, Belgium). A polypropylene glycol standard (MW ) 1200) was used (Waters, Milford, MA). A refinery gas-oil was provided by CEPSA (Madrid, Spain). TLC and Scanning Fluorescence Densitometry. Silica gel plates (aluminum sheets, 20 × 20 cm; 5-25 µm particle size; 60 Å pore size; 0.2 mm layer; from Panreac, Barcelona, Spain), and silica gel-impregnated (coated) plates were used. Berberine sulfate and 2,4,5,7-tetranitro-9-fluorenone (Across Chimica) were used as impregnating reagents. In both cases, a solution of the corresponding compound in methanol (2-6 mg × 100 mL-1) was used for impregnating during 20 s. These conditions provided adequate sensitivity in the subsequent detection. Plates were subsequently dried overnight at 40 °C. (22) Spartan version 5.0; Wavefunction, Inc. 18401 Von Karman Avenue, Suite 370, Irvine, CA 92612. (23) (a) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Chio, C.; Alacona, G.; Profeta, S.; Weiner, P. J. Am. Chem. Soc. 1984, 106, 765. (b) Weiner, S. J.; Kollman, P. A.; Nguyen, D. T.; Case, D. A. J. Comput Chem. 1986, 7, 230. (24) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440. (25) Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989, 111, 4379.

Figure 3. Fluorescent chromatographic responses of pure nalkanes with sample load: A, n-C12; B, n-C16; C, n-C18; D, n-C24; E, n-C30. (Development, 9 min with n-hexane; impregnation, 2 mg berberine/100 mL of methanol; volume application, 0.4 µL; beam size, 1 × 1 mm).

After application, alkanes were developed during 9 min using n-hexane. The other compounds were detected by fluorescence scanning densitometry, either after development using the same conditions as alkanes or without development, at the application point. A CS9301 TLC scanner (Shimadzu, Tokyo, Japan) was used in the fluorescence mode (λexc ) 365 nm; filter 3; linear scanning; typical beam size, 1 × 1 mm) to provide detection of the corresponding compound. Peak positions and distances of spotting between the different lanes were determined by using conventional scanning (perpendicular to the direction of development, at the y-coordinate corresponding to each developed peak). Peak area data were taken from the scanner and all data were collected, displayed, and stored using Shimadzu CS9310 PC software. Data output was linearized in the densitometer using the Kubelka-Munk procedure.26

RESULTS AND DISCUSSION Figure 2. TLC chromatograms of some compounds determined by enhancement of fluorescence response using berberine-coated silica gel plates: (A, B) n-C24 and n-C16 (different sample loads), respectively (both eluted 9 min with n-hexane); (C) from left to right, geraniol (3 µg), galactocerebroside (3 µg), prostaglandin VII (3 µg), cholesterol (2.1 µg), cholestane (2.7 µg), and ceramide (1.6 µg) (without eluting, at the application point); (D) refinery gas-oil (boldface chromatogram, saturates by fluorescence; lightface chromatogram, aromatics by ultraviolet).

Commercially available caffeine-coated silica gel HPTLC plates (Nano-Sil-PAH; glass plates, 20 × 10 cm; 2-10 µm particle size; 60 Å; 0.2 mm layer) from Macherey-Nagel (Du¨ren, Germany) were also used. Samples were spotted onto TLC plates using an SES 3202/IS02 automatic spotter (Bechenheim, Germany). Volumes applied were usually between 0.4 and 1 µL of samples. Sample loads ranged from 400 ng to several micrograms. Samples were injected a minimum of three times.

Fluorescence Enhancement Involves Alkane-Berberine Interaction. Alkanes have classically been considered as inert molecules. They do not exhibit fluorescence or ultraviolet spectra within the usual wavelength range. However, when silica gel plates are impregnated with berberine (a fluorescent alkaloid, in the form of hydrochloride or sulfate), the presence of paraffinic compounds produces an increase in the fluorescence signal when sample is irradiated with monochromatic UV light (e.g., at λ ) 365 nm). Figure 1 shows the variation of the fluorescent chromatographic response (in area counts) with the excitation wavelength of the n-C16 spotted on a berberine-impregnated plate. Figure 2 shows chromatograms corresponding to several samples studied in this work, which illustrate the phenomenon of fluorescence enhancement produced by berberine: Figure 2A and B shows the possibility of quantifying n-alkanes after elution. Figure 2C shows several examples of molecules other than n-alkanes that have been detected, in this case, without elution at the application point. Their responses are studied later on in this (26) Kubelka, P.; Munk, F. Z. Tech. Phys. 1931, 12, 593.

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Figure 4. Chief geometric data of berberine (A) and berberine-n-pentane complex (B) in their ground state (HF/PM3 level, boldface numbers) and first excited state (3 × 3CI-HE/PM3 level, lightface numbers). Bond distances are given in angstroms. The different atoms are represented by increasing order of shading, as follows: H, C, O, N.

work. Finally, Figure 2D illustrates the detection of saturated compounds in a real matrix (gas-oil) by fluorescence scanning densitometry and the aromatic fraction determined using UV scanning densitometry (λ ) 250 nm) of the studied samples. More details on the application of the phenomenon of fluorescence enhancement to petrochemical analysis for quantitatively determining hydrocarbon types in petroleum products can be found elsewhere.27 Thus, hydrocarbon types have been successfully determined in middle (gas-oil) and heavy (lubricants, heavy oil, vis-breaking fuel) petroleum distillates with adequate precision, and quantitative results agree with those provided using other well-established techniques in the petrochemical industry. The sensitivity of this analysis has been tailored through control of berberine concentration and impregnating time. (27) Cebolla, V. L.; Membrado, L.; Domingo, M. P.; Henrion, P.; Garriga, R.; Gonza´lez, P.; Cossı´o, F. P.; Arrieta, A.; Vela, J. J. Chromatogr. Sci. 1999, 37, 219.

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For a given wavelength, increase in fluorescence signal is proportional to sample load for a given alkane and is also dependent on the alkane structure, as shown in Figure 3. In the case of the n-alkane series, the longer the chain, the greater the area of the chromatographic peak. Under the impregnation conditions used in this case, C12 provided a weak signal. It is to be noted that the addition of each new CH2 group seems to give an increase in response. To shed some light on the reasons underlying the enhancement in the fluorescence signal, we first performed a complete geometry optimization of the berberine cation. Given the size of this structure, these calculations were carried out using the PM3 semiempirical Hamiltonian16 at the HF level, since this method has proven to yield reliable geometries in many nitrogencontaining heterocyclic compounds and cations.28 On the basis of spectral measurements in solution5 (vide supra), sorbentberberine or sorbent-alkane interactions were not included at

Table 1. Averaged Complexation Energies (∆E) for Different Ion-Molecule Structures As Computed by Means of the AMBER Force Field

Figure 5. Canonical frontier orbitals of berberine at HF/PM3 level: A, HOMO; B, LUMO.

complex

∆E (kcal mol-1)

berberine-C12 berberine-C14 berberine-ceramide berberine-galactocerebroside

-7.04 -7.50 -18.76 -16.63

HE level, the fluorescence wavelengths of the S1 f S0 transition are found to be 506 nm for berberine and 474 nm for the berberine-n-pentane complex. This corresponds to Stokes shift values of 80 and 57 nm, respectively. According to these results, the enhancement of intensity observed in the berberine-alkane systems can be explained by considering two factors. According to classic quantum theory,30 the intensity of the emitted signal is proportional to the Einstein coefficient of spontaneous emission A: 2

this stage of our computational analysis. The chief geometrical features of berberine cation at the HF/PM3 level in its ground state are reported in Figure 4A (boldface numbers). From these data, it can be readily concluded that berberine is a nearly planar cation with a remarkable conformational rigidity. This fact precludes explanations on the berberine-alkane interactions based upon conformational changes, as has been proposed for other systems.28,29 The frontier molecular orbitals of berberine cation are displayed in Figure 5. The HOMO is located mainly on the D ring, whereas the larger contributions to the LUMO correspond to the B ring. Therefore, an electronic excitation involves a reorganization of the electron density in the whole molecule. We also optimized, at the HF/PM3 level, the ionmolecule complex between n-pentane and the berberine cation. n-Pentane has been used for simplification of calculations. The structure of this complex is drawn in Figure 4B. Our calculations suggest that there is a weak electrostatic interaction between the alkane molecule and the electron-deficient π-system of berberine and that there are intermolecular distances close to van der Waals contacts in several positions. Our results also indicate that the composition of the frontier molecular orbitals is not affected by the interaction with the alkane. According to our calculations, the wavelength associated with the HOMO-LUMO excitation is 426 nm for berberine and 417 nm for the berberine-pentane complex, in the gas phase. We next optimized the geometries of berberine and the berberine-n-pentane complex in the first excited state at the 3 × 3CI-HE/PM3 level of theory. The chief geometric data are given in parts A and B of Figure 4 (lightface numbers), respectively. As can be seen, the S0 f S1 transition induces considerable variation in the berberine geometry in both cases, mainly because of the antibonding C-N interaction in the B ring. At the 3 × 3CI(28) (a) Buterbaugh, J. S.; Toscano, J. P.; Weaver, W. L.; Gord, J. R.; Hadad, C. M.; Gustafson, T. L.; Platz, M. S. J. Am. Chem. Soc. 1997, 119, 3580. (b) Parusel, A. B. J.; Schamschule, R.; Piorun, D.; Rechthaler, K.; Puchala, A.; Rasala, K.; Rotkiewicz, K.; Ko¨hler, G. J. Mol. Struct. (THEOCHEM) 1997, 419, 63. (29) DiCe´sare, N.; Belleteˆte, M.; Raymond, F.; Leclerc, M.; Durocher, G. J. Phys. Chem. A 1998, 102, 2700.

A ) (8π2νfi /3pc2)µfi

2

(1)

where vfi is the transition frequency, p is h/2π, c is the speed of light, µfi is the transition dipole operating over the final and initial wave functions,

µfi ) 〈ψf|M|ψi〉 )

∫ ψ µψ dτ * f

i

(2)

and  is the dielectric constant of the medium. Therefore, the polar environment provided by the silica gel induces a lowering in the fluorescence intensity. A lipophilic compound that interacts with berberine and surrounds it creates an apolar microenvironment that, in turn, lowers the dielectric permittivity, thus enhancing the intensity of the fluorescence signal. On the other hand, this intensity also depends on the quantum yield Φ,31 given by

Φ ) Γ/(Γ + k)

(3)

where Γ is the emissive rate of the fluorophore and k stands for the grouped rate constants of all possible nonradiative decay processes. According to eq 3, the second effect of the lipophilic compound surrounding berberine is to hinder the efficiency of these nonradiative transitions, thus enhancing the quantum yield and consequently the intensity of the fluorescence signal. In summary, our computational analysis suggests that the role of the alkane is to provide an apolar environment to the excited berberine cation, which hinders alternative relaxation mechanisms and favors the fluorescence emission. We also optimized the geometries of two model berberinealkane complexes (using n-C12 and n-C14) by means of the AMBER force field.23 Given the size of the structures, the optimal conformations were located using Monte Carlo simulations.25 The (30) Atkins, P. W.; Friedman, R. S. Molecular Quantum Mechanics; Oxford University Press: Oxford, U.K.,1997; p 508. (31) Lakowitz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; pp 9-11.

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Figure 6. Lowest energy conformations (see text) of complexes between berberine and: A, n-C12; B, n-C14; C, ceramide; D, galactocerebroside. Atoms are represented as follows: H, white; C, gray; O, red; N, blue.

resulting interaction energies are reported in Table 1 and the corresponding geometries are shown in Figure 6A,B. No significant discrepancies were observed between the berberine geometries computed at the RHF/PM3 and AMBER levels. Our results show a considerable conformational rigidity in berberine, thus indicating that the response for the different analytes must not be due to significant changes in the berberine geometry on passing from one alkane to another. As can be seen from Figures 4 and 6A,B, the alkane adopts an extended conformation along the main axis of the berberine molecule in order to maximize the ion-molecule interaction. Our results also show comparable but slighty higher ∆E values for n-C14 with respect to n-C12, in good agreement with our experimentally found fluorescence intensity enhancements for different alkanes (vide supra), since, according to our models, the larger ∆E values provide a more consistent apolar environment to the berberine molecule. 1764 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

Analytes Other Than Alkanes. Apart from paraffinic compounds, the theoretical study suggests the possibility of using the berberine cation to detect other compounds with partially saturated structure (Figure 2). Among the many possible structures, Figure 7 shows structures that we have successfully detected using berberine cation (1): stearic acid (2), oleic acid (3), 5Rcholestane (4), 5β-cholestan-3R-ol (5), cholesterol (6), stearylcholesterol (7), prostaglandin VII (8), a ceramide (9), a galactocerebroside (10), geraniol (11), and a propylene glycol (12). Table 2 shows response factors for some of these compounds obtained under the same conditions. Compounds 8-12 showed lower fluorescence responses than compounds 2-7. According to eqs 1-3, this may be explained by taking into account the higher polarity of analytes 8-12. The more saturated the compound, the lower the polarity and therefore, the higher the fluorescent response. The low response of prostaglandin VII is

Table 3. Different Responses (Area Counts per Mass Unit, µg) for n-C24 on Berberine, Caffeine, and Tetranitrofluorenone-Coated Plates (λexc ) 365 nm) analyte-impregnant n-C24-berberinea n-C24-caffeineb

n-C24-tetranitrofluorenoneb

A/m 1168 6.5 8.5

a n-C was developed 9 min in n-hexane. b n-C was determined at 24 24 the application point.

Figure 7. Chemical structures of berberine cation and the compounds detected by fluorescence enhancement (see text). Table 2. Response Factors (Area Counts per Mass Unit, µg) of Some Apolar and Polar Analytesa on Berberine-Coated Platesb analyte

A/m

5,R-cholestane galactocerebroside polypropylene glycol geraniol prostaglandin VII

722 343 274 350 347

a n-C , estearic acid, and estearyl cholesterol saturated the detector 24 under these conditions. b A 3 µg aliquot of each compound determined at the application point; berberine concentration, 6 mg/100 mL of methanol.

due to its polar-aromatic nature. Other prostaglandins with more saturated structures would be better detected using berberinecoated plates. We have selected the berberine-ceramide and berberinegalactocerebroside complexes (Figure 6C and D, respectively) as model structures for this kind of analyte. Our results indicate that these preferentially adopt conformations in which the apolar

chains are over the apolar rings of berberine and the polar hydroxy groups are very close to the positively charged nitrogen atom of berberine. This results in larger ∆E values for these later complexes (see Table 1). However, given the polar nature of these latter interactions and the nature of the S1 f S0 transition of berberine (vide supra), the polar environment around the nitrogen atom induces a lower intensity enhancement because of the larger value of the  and k terms with respect to the berberine-alkane structures. Planar, Ionic, and π-Accepting Systems Are Required for Fluorescence Enhancement. Other rigid, planar, π-acceptor molecules (caffeine and tetranitrofluorenone, TNF) have been studied as impregnation reagents. Although both interact with alkanes and give fluorescence enhancement, the chromatographic response is ∼150 times lower than in the case of berberine (Table 3). Therefore, π-acceptor systems are a necessary condition for fluorescence enhancement but are not sufficient on their own. This is in agreement with the proposed explanation: berberine is a cation and its interaction with alkanes is stronger than in the case of uncharged acceptor caffeine or TNF. Thus, the apolar microenvironment that improves the quantum yield and enhances the Einstein coefficient A of the fluorescent relaxation may be less efficient and there may be microaggregates of caffeine (or TNF) that would keep the alkanes away and would relax by other polar ways. CONCLUSIONS The analytical phenomenon of alkane-mediated enhancement of fluorescence is reported in this work, and an original explanation is proposed for taking into account the experimental data. Computational calculations, based on molecular orbital theory and molecular mechanics, suggest that an analyte-berberine interaction is responsible for this phenomenon. This is the first time that an explanation based on molecular interactions involving an n-alkane is proposed for the fluorescence enhancement. The resulting fluorescence signal is useful for analytical purposes. The role of the alkane is to provide an apolar environment for the excited berberine cation which lowers the dielectric constant around berberine and hinders alternative relaxation mechanisms, thus favoring fluorescence emission. This explanation has allowed new interacting systems of analytical interest to be proposed, which have been experimentally confirmed. Not only alkanes but other compounds with a partially aliphatic structure give an increase in fluorescence signal when the berberine cation-analyte system is irradiated with UV light. This phenomenon may be of interest for determining compounds Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

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with a low UV response, in the environmental field, petrochemistry, and polymer science and in the determination of some compounds with biological interest.

edges a grant from the Agence Franc¸aise pour la Maıˆtrise de l’Energie (ADEME, France), and from the Rotary Club (Hagondange, France).

ACKNOWLEDGMENT This work was supported by the Plan Nacional de I+D (Spanish CICYT, project QUI98-0852), the Gobierno Vasco/Eusko Jaurlaritza (project GV 170.215-EX97/11). P.H. gratefully acknowl-

Received for review November 17, 1999. Accepted January 18, 2000.

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AC991302Q