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Anal. Chem. 1990, 62, 1144-1147
Investigation of the Microenvironment Polarity of a Chromatographic Surface Using Total Internal Reflection Fluorescence Vinay M. Rangnekar and Philip B. Oldham* Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762
A planar chrmatographlc &ace was etwlled for microenvhonmental polarity by using total Internal refle&on fluorescence (TIRF). Pyrene was ad8ofbd on two dmerenl chemically modHled quartz plates. The surfaces of the mocNfled quartz plates were covered wlth elther a monomerlc Cl0 or a polymeric C,* layer. TIRF data were obtalned for the adsorbed pyrene In the presence of dmerent polar solvents. As the polarity of the solvents Increased, an overall Increase in fluorescence intensity was observed and a change In the relative vlkonlc bend Wenrilles (III/I) of pyrene was noted. Also, varlable-angle TIRF data were collected for the adsorbed pyrene In the presence of a 40% methanol/water mixture (v/v). The fluorescence contrlbutlon from the bulk fluorescence was found to decrease as the Incidence angle increased, as was expected.
INTRODUCTION Liquid chromatography is one of the most commonly used methods of chemical separations. The extent of separation depends primarily on solute-solvent and solute-stationary phase interactions. A thorough understanding of these interactions largely depends on the chemical and physical nature of the stationary phase ( 1 , 2 ) . In the last decade, a considerable amount of work has been directed toward the characterization of chromatographic stationary phases. Of particular interest are the hydrophobic bonded phases commonly used in reversed-phase liquid chromatography. Separation effectiveness is largely determined by the distribution and conformation of the bonded phase. A number of spectroscopic methods have been employed to determine the stationaryphase polarity (3,4)and viscosity (5),the association between alkyl chains (61, and the degree of chain extension (7). In particular, the sensitivity of fluorescence measurements to the microenvironment of a probe molecule has been shown to provide considerable information concerning the stationaryphase environment (3-12). Microenvironment polarity and viscosity have been determined by the examination of spectral wavelength shifts and the excited-state lifetimes of the fluorescent solutes in the bulk solution (2-4,12).However, most of the investigations performed were unable to observe the chromatographic interactions while in progress, since the experimentswere limited to static chromatographic conditions. The ability to probe solid/liquid interfaces by the surface-selective excitation of fluorescencewas first proposed by Hinchfeld (13).Total internal reflection fluorescence (TIRF) is a highly sensitive interfacial technique capable of detecting fluorescent molecules within a few hundred nanometers of a surface (interface). This paper demonstrates the utility of TIRF for the investigation of a planar chromatographic surface. THEORY The underlying principles of TIRF are well documented in the literature (14-20). In brief, the penetration depth of 0003-2700/90/0362-1144$02.50/0
the exponentially decaying evanescent wave into the less dense medium is given by d(ei) = &/47f[(nl sin 6J2 - n3-1/2 (1) where ei is the incidence angle (greater than the critical angle), Xi is the incident light wavelength, and nl and n2 are the refractive indices of the two media (nl > nz). In TIRF the evanescent wave is primarily responsible for fluorescence excitation. An important aspect of TIRF involves the reverse coupling of the emitted fluorescence back across the interface into the optical waveguide. The effect of the observation angle on this reverse coupling is similar to the effect of the incidence angle on the evanescent wave penetration depth. Therefore, the TIRF data can be collected a t a fixed observation angle for varying incident angle or at a fixed incident angle for varying observation angle. This variable-angle technique (20) is referred to as variable-angle total internal reflection fluorescence (VA-TIRF). The fluorescence detected by the VA-TIRF technique is given by (20)
If ~ [ t ( ~ i ) 1 2 [ ~ ( ~ , exd-z/[Wi) ) 1 2 ~ m ~ ( ~+ )4e0)l) dz (2) where K = kikot41i, is the extinction coefficient, 4 is the fluorescencequantum efficiency of the fluorophore, ki and KO are proportionality constants, and Ii is the incident light intensity. The terms in eq 2 are as follows: Ifis the fluorescence intensity; [t(ei)I2and [t(0,)]2 are the Fresnel transmission coefficients (21) for the incidence and the observation angle, respectively; c ( z ) is the concentration of the fluorescing solution at a distance ( z ) from the interface; and d(@ and d(0,) are the penetration depths of the evanescent wave for the incidence and the observation angle, respectively. Literature reports indicate the utilization of VA-TIRF to study fluorescence density profiles of Langmuir-Blodgett deposited thin films on flat surfaces (22) and the monomer concentration profile of an interfacial layer of polymer solutions near a flat, transparent, solid wall (23-25). The VATIRF data (eq 2) are a convolution of the fluorophore concentration-depth profile and the decaying evanescent wave used for excitation. VA-TIRF could potentially provide a mechanism for calculating the density of fluorescent solute, with respect to the distance away from the solid support, in a chromatographic system. The sensitivity of TIRF to localized interfacial dynamics makes it an ideal technique for intimately studying the mechanisms of soluteaolvent-stationary phase interactions. The main objective of this study was to explore the potential of TIRF for the investigation of the microenvironment of a chromatographic surface using pyrene as a fluorescent probe.
EXPERIMENTAL SECTION Materials. Spectrophotometricgrade methanol purchased from Fisher Scientific (FairLawn,NJ) and pyrene obtained from Aldrich Chemical Co., Inc. (Milwaukee,WI)were used as received. Octadecyltrialkoxysilane (Aquasil) was purchased from Pierce (Rockford,IL), and chlorodimethyloctadecylsilane(Cl-ODS)was 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62,NO. 11, JUNE 1, 1990 [Ex
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purchased from Petrarch Systems (Bristol, PA). Both reagents were used as received. A 2 X lob M solution of pyrene in methanol was prepared. Also, methanol/water mixtures were prepared containing80%, 60%,50%, and 40% methanol by volume. Prior to each experiment the test solutions were purged with nitrogen gas to minimize oxygen quenching. Equipment. The fluorescence spectra were acquired on a Photon Technology International Inc. (Princeton, NJ) spectrofluorometer. The W-visible source was a 75-W xenon short-arc lamp (Ushio Inc.). A R928P type photomultiplier tube (Hamamatsu Photonics K.K.) was used as the detector. For the TIRF measurements the sample compartment of the spectrofluorometer could not be modified so as to accommodatethe necessary TIRF optics. Therefore, a new sample compartment of dimensions 30 X 30 X 20 cm was constructed. Horizontal and vertical translational stages purchased from J. A. No11 Co. (Monroeville,PA) were installed for easy alignment of an internal reflection assembly (TMP-320) purchased from Harrick Scientific (Ossining, NY). A single-pass parallelepiped 60" quartz plate of dimensions 50 X 20 X 3 mm also purchased from Harrick served as the waveguide. A diagram of the optical configuration is presented in Figure 1. The quartz plate surfaces were chemically modified with CLODS or Aquasil according to the procedure described earlier (26). The CLODS forms a monomeric phase on the quartz surface, while the Aquasil forms a polymeric Cla phase. Light from the excitation monochromator (330nm) was focused with a fused silica lens of focal length 25 mm on one edge of the quartz plate at an incidence angle greater than the critical angle (65"). The quartz plate was placed in a liquid flow cell (Harrick) containing pyrene solution. The multiple internal reflections (number of reflections = 7) produced an evanescent wave, which excited the near-surface pyrene molecules. The fluorescence generated was coupled back into the quartz crystal and collected from the opposite end of the crystal by using the two mirrors of the internal reflection assembly, and a fused silica lens of focal length 25 mm was used to focus the emitted light onto the entrance slit of the emission monochromator. The horizontal and vertical stages were adjusted so as to obtain maximum sensitivitywithout spectral distortion. Procedure. The c18 surface modified quartz plate was mounted in the cell containing the 2 X M pyrene solution. Thii pyrene solution was allowed to stand in the cell for 5-10 min. During this period, pyrene adsorbed onto the cl8 phase. Later the pyrene solution was removed, and the cell was washed with deionized water. As the solubility of pyrene in water is considered negligible, the adsorbed pyrene remains on the quartz plate. Next methanol was introduced into the cell, and TIRF data were collected. The cell, along with the quartz plate, was later thoroughly washed with methanol to remove any adsorbed pyrene. The above-mentioned procedure was repeated to examine the behavior of solvent polarity (loo%,SO%, 70%,60%,40%, and 0% by volume MeOH/water solution) on adsorbed pyrene. The incidence angle (ei) for the TIRF data collection was 67". TIRF data were also collected for the pyrene adsorbed on a monomeric
W A V E L E N G T H
Figure 2. Solvent effects on pyrene adsorbed on an Aquasll-modified
quartz plate (excitation wavelength 330 nm, emission wavelength 360-460 nm). [Ex = 330 nm : Em
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quartz plate (excitation wavelength 330 nm, emission wavelength 360-460 nm). as well as a polymeric C18phase in the presence of a 40% (v/v) MeOH/water mixture at different incidence angles, viz. 67", 70°, 7 4 O , 77", and SOo.
RESULTS AND DISCUSSION Generally, pyrene exhibits five vibronic bands in the region 375-400 nm. Due to the moderate resolution of the instrument, only bands I (376 nm), I11 (385 nm), and V (397 nm) could be observed. The ratio of intensities of bands III/I can be used to study the microenvironmental changes. An increase in microenvironmental polarity should decrease the III/I ratio (27). The III/I ratios, calculated from the bulk fluorescence data and the TIRF data collected in our laboratory, were in close agreement with those reported in the literature (27). The emission spectra (360-460 nm) obtained for pyrene adsorbed on both the polymeric Cls stationary phase and the monomeric C18phase in the presence of different solvents are presented in Figures 2 and 3, respectively. The fluorescence intensity ( I f )observed is a combination of the fluorescence contribution from the pyrene in the bulk solution and pyrene adsorbed on the Cls phase. In general, as the percent MeOH decreases, the I f increases and also changes in the relative vibronic band intensities (III/I) are observed. Since pyrene is insoluble in water, the amount of adsorbed pyrene reequilibrating in the MeOH/H20 mixture decreases as the percent water increases. Thus, the amount of pyrene at the interface is greater and the overall fluorescence intensity increases. When the pyrene is associated with the stationary
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
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Wnetration Depth (nm) Figure 7. Plot of pyrene III/I band intensity ratio versus penetration depth.
phase, it experiences a nonpolar environment and affects the III/I band ratio. In the case of pyrene adsorbed on the polymeric phase, the calculated relative band intensities (III/I) are found to increase slightly as the percent methanol increases (40%-100% MeOH) (Figure 4). This indicates an increasing nonpolar environment experienced by the pyrene. Considering the insoluble nature of pyrene in water, one would expect an increasing association of the pyrene to the stationary phase with the decreasing percent MeOH. However, the observed effect may be due to the fact that the bulk fluorescence contribution is still too significant compared to the surface fluorescence and so the pyrene spectra appear to represent a more polar environment than expected at low percent MeOH. For pyrene adsorbed on the monomeric phase, the calculated relative band intensities (III/I) are found to generally decrease gradually as the percent methanol increases (Figure 4). This suggests that the pyrene is in the nonpolar environment as expected a t low percent MeOH solvent composition. The discrepancy in the two sets of data suggests that either pyrene has more affinity for the monomeric phase than for the polymeric phase, which would seem unlikely, or the surface coverage of the monomeric phase is greater than that of the polymeric phase. In the presence of 100% water, the adsorbed pyrene is trapped into the Cla phase, as pyrene in insoluble in water. Hence, the relative band intensity (III/I) is greater than one, suggesting a nonpolar environment for the pyrene. However, it is important to note that these data are very preliminary and sufficient reproducibility data must be acquired to verify these observations. Future studies are being
planned that utilize a peristaltic pump and flow cell assembly to facilitate convenient changeover between solvents. The VA-TIRF measurements a t different incidence angles 67", 70°, 74", 77O, and 80" for the pyrene adsorbed on the monomeric as well as the polymeric CI8phase are shown in Figures 5 and 6, respectively. The penetration depth of the evanescent wave decreases as the incidence angle increases as per eq 1. For the angles selected, the penetration depth ranged from approximately 90 to 220 nm. Hence, the Ifdecreases with the increase in the incidence angle due to reduced sample volume. The calculated relative band intensities (III/I) are found to increase with increasing incidence angle (Figure 7). This is due to the fact that as the incidence angle increases, the fluorescencecontribution from the bulk solution decreases and ideally only the near-surface pyrene which is in the nonpolar environment is detected. For comparison, the fluorescence contribution from bulk solution is approximately 89% at ei = 67", which drops to 77% at Bi = 80". Because of the limited movement of the internal reflection assembly and the 60" beveled angle of the internal reflection crystal, we are currently limited to Bi 5 80". Theoretical calculations show that at Bi = 89" the bulk fluorescence contribution is approximately66%. Another, important point to note is that these values are first approximations with respect to the excitation efficiency of the evanescent wave only and the collection efficiency was not taken into account. A plot of the normalized relative intensities of bands I and I11 versus the incidence angle (Figure 8) contains information regarding the distribution profile of the pyrene molecules as a function of depth in solution. The true concentration-depth
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990 1.0
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concentration-depth profile. There is a potential for extracting the solute concentration-depth profile from the VA-TIRF data, which should help answer the question as to where solute retention occurs, on the interfacial surface or withh the stationary phase. The degree of CI8chain extension and association needs also to be examined to get a better understanding of the behavior of the stationary phase in different solvent environments. Registry No. MeOH, 67-56-1;pyrene, 129-00-0;vitreous silica, 60676-86-0; octadecylsilane, 18623-11-5; chlorodimethyloctadecylsilane, 18643-08-8.
LITERATURE CITED
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profile can be potentially extracted by performing an inverse Laplace transform (20) of eq 2. However, such a procedure is difficult, particularly in the presence of noise. Another method is to calculate the fluorescence intensity by using eq 2 and assuming various model concentration profiles and then to use the least-squares criteria to determine the goodness of fit between the calculated and experimental data. Additional experiments in this area are currently underway. In conclusion, TIRF is a promising technique to examine a chromatographic surface. The optical configuration increases the sensitivity due to the multiple internal reflections, which promote multiple excitation-sample interactions. The TIRF data obtained exhibit moderate spectral resolution, which could certainly be improved by using a higher resolution monochromator. The use of a very intense and collimated source (i.e., laser) is not essential for TIRF measurements. Hence, the use of a conventional fluorometer for internal reflection fluorescence studies has been demonstrated. Distinction between the fluorescence contribution from the bulk solution and the interfacial surface can be easily made by the TIRF technique. The VA-TIRF data suggest the observation of near-surface fluorescence at higher incidence angles. The unique ability to control the depth of penetration of the evanescent wave into the chromatographic system provides data that contain information about the solute
(1) Gllpln, R. K. Anal. chem. 1985, 57, 1465A. (2) Sander, L. C.; Callis, J. 8.; Field, L. R. Anal. Chem. 1983, 55. 1068. (3) Lochmuller, C. H.; Marshall, D. 6.; Wllder. D. R. Anal. Chlm. Acta 1981, 730, 31. (4) Stahiberg, J.; Almgren, M. Anel. Chem. 1985, 57. 817. (5) Stahlberg, J.; Almgren, M.; Alsins, J. Anal. Chem. 1988, 80, 2487. (6) Lochmuiler, C. H.; Colburn, A. S.; Hunnlcut. M. L.; Harris, J. M. Anal. Chem. 1983, 55, 1344. (7) Gilpin, R. K.; Kasturi, A.; Gelerinter. E. Anal. Chem. 1987. 5 9 , 1177. (8) . . Lochmuiler. C. H.: Marshall. D. E.: Harris. J. M. Anal. Chlm. Acta 1981, 737, 263. (9) Doug, D. C.; Wlnnik. M. W. Photochem. Phot06lol. 1984, 35, 17. (10) Carr, J. W.; Harris, J. M. Anal. Chem. 1988, 56, 626. (11) Carr, J. W.; Harris, J. M. Anal. Chem. 1987, 5 9 , 2546. (12) Hartner, K. C.; Carr, J. W.; Harris, J. M. Appl. Spectrosc. 1989, 43, 81. (13) nrschfeld, T. Can. J . Spectrosc. 1985, IO. 128. (14) Harrick, N. J. Internal Reflecflon Spectroscopy; Interscience: New York, 1967. (15) Axelrod, D.; Burghardt, T. P.; Thompson, N. L. Annu. Rev. Blophys. Bioeng. 1984, 73,247. (16) Rockhold, S. A.; Qulnn, R. D.; VanWagenene, R. A.; Andrade, J. D.; Reichert, M. J. J . Electroanal. Chem. 1983, 150, 261. (17) Harrick, N. J.; Loeb, G. I.Anal. Chem. 1973, 45, 887. (18) Place, J. F.; Sutherland, R. M.; Dahne, C. Blosensws 1985, 7 , 321. (19) Harrick, N. J.; Loeb. G. I. I n Modern Fluorescence Spectroscopy; Wehry, E. L., Ed.; Plenum: New York, 1976; Vol. 1. (20) Reichert, W. A.; Sucl. P. A.; Ives, J. T.; Andrade, J. D. Appl. Spectrosc. 1987, 47, 503. (21) Lee. El-Hang; Benner. R. E.; Fenn. J. 6.: C h a m R. K. ADD/. . . O.D ~ . 1979, 78, 832. (22) Suci, P. A.; Reichert, W. M. Lanqmuir 1988, 4 , 1131. (23) Ailains, C.; Ausserre, D.; Rondelez. F. phvs. Rev. Lett. 1982. 49. 1694. (24) Ausserre, D.; Hervet, H.; Rondelez, F. Phys. Rev. Lett. 1985, 5 4 , 1948. (25) Ausserre, D.; Hervet, H.; Rondelez, F. Mecromo/ecules 1988, 79, 85. (26) Rangnekar, V. M.; Oldham, P. B. Spectrosc. Left. 1989, 22, 993. (27) Dong, D. C.; Winnlk, M. A. Photochem. Photoblol. 1982, 35. 17.
RECEIVED for review December 27,1989. Accepted February 26, 1990. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for the support of this research. P.B.O. also acknowledges support from the National Science Foundation (Grant RII-8902064) EPSCoR program.
