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Anal. Chem. 1990, 62, 7438-7444
potential and membrane resistance, depending on their chemical structures. These phenomena were well correlated with the behavior of bitter taste or olfactory reception on gustatory or olfactory cells in humans. Such simple membrane system may provide useful models of chemoreceptors in biological membranes.
LITERATURE CITED (1) Ammore, J. E. Molecular Basis of Odor; Thomas: Springerfieid. IL. 1970. (2) Kurihara, K.; Yoshii, K.; Kashiwayanagi, M. Comp. Biochem. Physiol., A : Comp. physiol. 1986, 85A, 1-22. (3) Price, S. Nature 1969, 227,779. (4) Tucker, D. Handbook of Sensory Physiology; Belidier. L. M., Ed.; Springer: Berlin, 1971; Vol. IV-1, pp 151-204. (5) Davies. J. T. Handbook of Sensory Physiology; Beiidier, L. M.; Ed.; Springer: Berlin, 1971; Voi. IV-1, pp 322-350. (6) Koyama, N.; Kwihara, K. Nature (London) 1972, 236, 402. (7) Nomura, T.; Kurlhara, K. Blochemisfry 1987, 26, 6135, 6141. (8) Kumazawa, T.; Nomura, T.; Kurihara, K. Biochemistry 1988, 27, 1239. (9) Preliminary reports, Okahata, Y.; Ebato, H.; Taguchi, K. J. Chem. SOC., Chem. Commun. 1987, 1363. Okahata, Y.; En-na, G. J. Chem. Soc., Chem. Commun. 1987, 1365. (10) For a review, GulibauR, G. G. Ion-Sel. Nectrode Rev. 1980, 2, 3. (11) Turnham, B. D.; Yee, L. K.; Luoma, G. A. Anal. Chem. 1965, 57, 2120. (12) N-Ngwainbi, J.; Foley, P. H.; Kuan, S. S.; Guiibault, G. G. J. Am. Chem. SOC.1986, 708,5444.
(13) Muramatsu, H.; Diko, J. M.; Tarniya, E.; Karube, I. Anal. Chem. 1987, 59, 2760. (14) Roederer, J. E.: Bastiians, G. J. Anat. Chem. 1983, 5 5 , 2333. (15) Ebersole, R. C.; Ward, M. D. J. Am. Chem. SOC. 1988, 170. 8623. (16) McCaffrey, R. R.; Bruckensteln, S.; Prasad, P. N. Langmuir 1986, 2, 228. (17) Thompson, M.; Arthur, C. L.; Dhaliwal. G. K. Anal. Chem. 1986, 58, 1206. (18) Okahata, Y.; Ariga, K. J. Chem. Soc., Chem. Commun. 1987, 1535. (19) Okahata, Y.;Ariga, K. Langmuir 1989. 5 , 1261. (20) Okahata, Y.; Ariga, K. Thin Solid Films 1989, 778, 465. (21) Okahata, Y.; Kimura, K.; Ariga, K. J. Am. Chem. SOC. 1989, 7 7 7 , 9190. (22) Okahata, Y.; Ebato, H. Anal. Chem. 1989, 67,2185. (23) Bruckenstein, S.; Shay, M. J. flectroanal. Chem. 1985, 788, 131. (24) Nomura, T.; Iijima, M. Anal. Chim. Acta 1981, 737,97. (25) Okahata, Y.; En-na, G. J. Phys. Chem. 1988, 92,4546. (26) Okahata. Y.: Taauchi. K.: Seki. T. J. Chem. Soc.. Chem. Commun. 1985, 1122. (27) Okahata, Y.; En-na, G.; Taguchi, K.; Seki, T. J. Am. Chem. SOC. 1985, 707,5300. (28) Okahata, Y.; Ebato, H.; Ye, X. J. Chem. SOC.,Chem. Commun. 1988. 1037. (29) Shibusawa, T. J. SOC.Dyers Colour. 1979, 95, 175. (30) Kumazawa, T.; Kashiwayanagi, M.; Kurihara, K. Brain Res. 1985, 333,27. (31) Harris, H.: Kalmus, H Ann Eugenics 1949, 75,24
RECEIVED for review November 9,1989. Accepted March 14, 1990.
Surface-Enhanced Resonance Raman Spectroscopy of Liquid Chromatographic Analytes on Thin-Layer Chromatographic Plates Steven A. Soper,’ Kenneth L. Ratzlaff, and Theodore Kuwana* T h e Center f o r Bioanalytical Research and t h e Department of Chemistry, T h e University of Kansas, Lawrence, Kansas 66046
Surface-enhancedresonance Raman spectroscopy (SERRS) was incorporated as a detector for a liquid chromatography (LC)-coupled thin-layer chromatography (TLC) system. I n thls SERRS/LC/TLC system, effluent from the LC system was deposited onto the surface of a solid supporting matrix (TLC plate) with a modified XY pbtter under computer control. An activated Ag sol was added to the plate (enhancing substrate) and then interrogated with a remote-sensing Raman spectrometer. Optical ftbers carrled the excitatbn light to the TLC plate and the scattered Raman radiation to the spectrometer for analysis. The dye, pararosaniline acetate, was found to give a itneaf SERRS signal over the concentratlon of 1 X loJ to 1 X lo-’ M range. The limit of detection was 750 fmol. The advantages of the LC/TLC system with SERRS detection, in comparison to conventional solution phase SERRS, is that the solid matrix prevents the contlnued aggregation of the Ag/dye cornpiex, thereby giving stable signals for extended periods of time. With the proper choice of matrix for the adsorption process, enhanced SERRS intenstties are observed. The analytes are stored,decoupHng the analysis from the LC flow. Acquisition of the SERRS spectrum can therefore be taken at whatever convenient time and long integration times can be used to improve the signal-to-noise ratio. Present address: Los Alamos N a t i o n a l Laboratories, MS-M888, Los Alamos, NM 87545.
INTRODUCTION The application of Raman spectroscopy to liquid chromatography (LC) offers several advantages such as molecular information from the spectrum and the absence of interfering water bands due to the small Raman cross section. The major difficulty associated with coupling Raman spectroscopy to LC analysis, however, is the inherent inefficiency of the Raman process. One method to overcome this inefficiency is the use of a laser with an emission line in resonance with an electronic transition of the analyte molecule; Le., resonance Raman spectroscopy (RRS). D’Orazio and Schimpf (1)reported the RRS detection of LC-separated azoxybenzenes. Sensitivities in the millimolar range were attained. Koizumi and Suzuki ( 2 ) used RRS a t 488 nm to detect amines which had been derivatized by dabsyl-C1 (4-(dimethylamino)azobenzene-4’sulfonyl chloride). Detection limits on the order of 1.5 ng were reported. Additionally, the entire RRS spectra of the derivatized amines could be obtained under stopped-flow conditions. The difficulty associated with utilizing RRS is that many analytes with electronic transitions in the visible region of the spectrum also fluoresce so that the weaker Raman bands may be obscured by the more intense fluorescent background. Also, the RRS spectrum may be dominated by bands from the chromophoric group. The method of surface-enhanced Raman spectroscopy (SEW) offers a viable approach to solving the aforementioned limitations. The large enhancement in the Raman signal with
0003-2700/90/0362-1438$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
SERS (3)gives detection limits far superior to those of RRS, even if the scatterer does not possess an electronic transition a t the laser wavelength. The fluorescent background associated with RRS is decreased in SERS due to the fluorescent quenching by adsorption of the scatterer to the enhancing substrate. The surprisingly large enhancement of SERS has been explained in terms of two principal theoretical models. The first model, called the electromagnetic enhancement model (4-13), is explained in terms of a very large increase in the electric field a t or near the surface of the dielectric microspheroids. This increase is attributed to the coupling of the incident electric field to the surface plasmons of the metal substrate of a particular morphology. The second model, coined the chemical enhancement model (14-19), ascribes the intense Raman signal to the direct chemisorption of the scatterer to the metal substrate. This chemisorption results in the formation of a charge transfer complex with electronic levels accessible to the visible excitation. Freeman (20) and Winefordner (21,22)have reported the use of Ag colloidal hydrosols as the enhancing substrate for LC detection with SERS. The colloid was added to the LC effluent postcolumn. Under stopped-flow conditions, the entire SERS spectrum could be obtained. The problem associated with this procedure is that the addition of the analyte to the sol induces aggregation of the colloidal particles, which, after extended periods, causes a decrease in the SERS intensity. The decrease in the signal intensity is a result of the broadening of the surface plasmon resonance, with a corresponding decrement in the electric field strength resulting from this resonance. Force (23)incorporated an Ag electrode for the SERS analysis of pyridine in flow injection analysis. It was necessary to modulate the potential of the Ag electrode for preconditioning prior to adsorption and desorption after SERS analysis so that subsequent LC components could be analyzed. Cotton and co-workers (24)have recently utilized SERS as an ancillary detector for high-performance liquid chromatography (HPLC). In their work, nitrophenols were separated on a reverse-phase HPLC column followed by collection of the LC fractions. A preroughened Ag electrode was introduced into the fractions with SERS spectra acquired after adsorption of the nitrophenols on the Ag surface. Detection limits on the order of 14 ppb were reported. Surface-enhanced resonance Raman spectroscopy (SERRS) is employed as the detection method in this study for the interrogation of LC analytes which have been transferred onto a thin-layer chromatography (TLC) plate. The coupling of LC to TLC with SERRS analysis offers several unique advantages such as the following: (1)The isolation of the LC analytes on a TLC plate allows the acquisition of complete SERRS spectra with conventional scanning spectrometers which would be otherwise difficult in a flowing LC liquid. (2) The properties of analytes in the adsorbed state may differ and have desirable spectroscopic features from that in solution. (3) The LC analytes fixed in a stable state on the TLC matrix allow analysis by more than one spectroscopic method. Although LC analytes on the TLC plate could be subjected to further separation, the work reported herein is restricted to the demonstration of the concept. Also, we had previously shown the advantage of transferring a LC analyte to a TLC plate for laser-induced fluorescence analysis (25). That is, the fluorescence quenching of lysine diderivatized with naphthalenedialdehyde was circumvented when the derivatized lysine was adsorbed onto the TLC plate. The ability to obtain SERS spectra on solid supports has been demonstrated by a number of investigators. Tran deposited several dyes, which had been added to a Ag colloidal hydrosol, onto various types of filter papers (26,27). He found
1439
that the signal intensity was dependent upon the method of preparing the Ag sol and the type of filter paper onto which the dye/sol mixture was deposited. The detection limit was 0.5 ng for the dye crystal violet. Along this same line, Laserna obtained SERS spectra for p-aminobenzoic acid, acridine, 9-aminoacridine, 2-aminoanthracene and aminoquinoline on filter paper that had been previously coated with a Ag sol solution (28). The SERS signal for 9-aminoacridine was found to decrease substantially with time, and after ca. 40 min was no longer observable. Sequaris and Koglin obtained SERS spectra of nucleic purine derivatives that had been deposited onto high-performance TLC (HPTLC) plates (29). The Ag sol was postdeposited onto the plates with detection limits on the order of 30 ng. In a slightly different approach, VoDinh and co-workers have deposited Teflon microspheres (200 nm diameter) onto TLC plates (30-34). Ag was then vapor-deposited over the monodispersed spheres, followed by the deposition of the scatterer. By use of this procedure, detection limits of 10 ng were reported for benzoic acid. We previously demonstrated that the supporting matrix, an aluminum oxide TLC plate, profoundly influenced the signal intensity and stability of SERRS (35). The aluminum oxide surface enhanced the SERRS' intensity while inhibiting the continued aggregation of the Ag sol after the scatterer (analyte) was added. Thus, stable SERRS spectra could be obtained for extended periods of time. The concept of coupling LC to TLC has been previously demonstrated for fluorescence (36-38),for fluorescence line narrowing (39), and for diffuse reflectance infrared spectroscopy (40,41). In this paper, the LC effluent was deposited onto a TLC plate with a modified XY digital plotter under computer control. The SERRS analysis of the eluant was made with a remote-sensing Raman spectrometer, which incorporated optical fibers to carry the laser excitation to the TLC plate and to collect the scattered radiation.
EXPERIMENTAL SECTION Reagents and Sol Preparation. The dye, pararosaniline acetate, was obtained from Aldrich (Milwaukee, WI) and was used as received. The mobile phase consisted of NANOpure H 2 0 (Sybron Barnstead, Boston, MA) and either MeOH or MeCN which were obtained from Fisher (Fairlawn, NJ). The TLC plates were coated with aluminum oxide (basic) (Alltech, Deerfield, IL). Silver nitrate and sodium citrate, used in preparation of the sols, were obtained from Aldrich. The Ag sols were prepared according to the published procedure of Lee and Meisel (42)and will be briefly described. To 500 mL of boiling NANOpure H20, 0.09 g of AgNOBwas added. With rapid stirring, 10 mL of a 1%solution of citrate was added to the boiling H20/AgN03solution over a period of ca.45 min. After the addition of the reducing agent, the sols were allowed to boil for an additional 60 min, and then cooled in ice water. The sols prepared in this fashion resulted in an optical absorption maximum of ca. 415 nm. Scanning electron micrographs of these sols indicated an average particle size of ca. 100 nm. Prior to use, the sols were activated with KCl (43). Instrumentation. A block diagram of the Raman optical and the LC/TLC system is shown in Figure 1. A Liconix Model 5000 Ar ion laser (Santa Clara, CA) provided the source of excitation. The 514.5-nm emission line of the Ar+ laser was isolated from the plasma lines with a discriminating band-pass filter (CWL = 514.5 nm and HBW = 10 nm) obtained from Omega Optical Co. (Brattleboro, VT). A power level of 100 mW at the TLC surface was used in all experiments. A x-y-z micropositioner mounted on the laser head was fitted with a microscope objective lens (lox) for focusing the radiation from the laser onto the optical fiber. A 400-pm silica optical fiber (General Fiber Optics, Cedar Grove, NJ) was situated on the micropositioner and carried the excitation light to the TLC plate. The terminus of this fiber was placed in a focusing beam probe (Oriel Corp., Stratford, CT). The probe consisted of a small metal cylinder containing two matched f/1.7 lenses. The larger axis of the elliptically shaped laser beam on
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY
15, 1990 ,deposited spot I
d
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Deposition pattern of the LC effluent on the TLC plate. Each circle corresponds to the deposition of effluent at that location on the TLC plate. The spots are arranged in columns, running the length of the TLC plate. Figure 3.
Figure 1. Block diagram of the LClTLC system with SERRS detection: FO1, excitation optical fiber; F 0 2 , collection optical fibers; TLC positioner, modified XY plotter; i, injector; pump, LC pump and syringe pump (used for the sol); Optics. signal coUection optics (detailed diagram shown in Figure 2A): Mono., double grating monochromator; Amp,
amplifier;Disc, discriminator:Photon Ct, photon counter; Microproc., microprocessor for controlling the monochromator.
Flguro 2. (A) Collection optics of the remote-sensing Raman spectrometer: FO, collection optical fibers (from the TLC positiiner); Notch, Raman notch filter. (E) Positioner (replaced the pen holder of the XY plotter): FO1, excitation optical fiber; F02, collection optical fibers.
the TLC surface was ca. 400 fim. The excitation fiber and beam focusing probe were mounted on a positioner as shown in Figure 2B. The angle of the excitation beam relative to the TLC surface was 45'. Three silica fibers (1 mm i.d., General Fiber Optics) served as the collection fibers. They were positioned in a triangular arrangement and placed in a metal collar. These fibers were then attached to the positioner (Figure 2B) with the fiber at an angle of 90' relative to the TLC surface. The collection fibers were placed ca. 2 mm above the TLC surface. The laser beam impinged the surface directly below these collection fibers. A diagram of the filtering and detection optics is shown in Figure 2A. The collection fibers were situated at the focal point of the collimating lens cfll.0 fused silica lens, Oriel Corp.) by a x-y-z positioner. The three fibers were arranged parallel to the entrance slits of the monochromator. The scattered radiation was then sent through a Raman notch filter (Omega Optical) with a rejection ratio of 5 X lo4 a t 514.5 nm. An additional Corning Long Pass colored glass filter (Esco Products, Inc., Oak Ridge, TN) was placed after the notch filter to further reject the Rayleigh line. The scattered radiation was focused onto the entrance slits Instruments SA, Inc., of a double grating monochromator (DH-10, Edison, NJ) with a plano-convex lens (Oriel Corp.) f-matched to the monochromator. A Model 1020-MS microprocessor (Instrumenta SA)controlled the wavelength drive of the monochromator. The slits of the monochromator were rued at 100 pm and resulted in spectral resolution of 20 cm-'. A noncooled Hamamatau R E 2 7
(Shimokanyo,Japan) was used with a photomultiplier tube (PMT) bias voltage of -950 V. The PMT output was analyzed with a photon counting system composed of a Model 1121A amplifier/discriminator and a Model 1109 photon counter (EG&G Princeton Applied Research, Princeton, NJ). Unless otherwise stated, a I-s photon integration time was used, resulting in a scan rate of 3.5 cm-'/s. The positioner shown in Figure 2B replaced the usual pen holder of a modified Tektronix Model 4662 XY plotter (Beaverton, OR) and served to deposit the LC effluent onto the TLC plate (through a Teflon tube mounted on the positioner, 500 pm id., positioned 1 mm from the TLC surface). In addition to the standard x-y operation via plotting commands directed through the computer serial port, a circuit was added to allow the plotter to be spatially positioned with high precision and resolution. The plotter's microprocessor logic was bypassed and discrete TTL pulses were directed to the microstepping logic of the stepper motors. The x-y plotter had a spatial resolution of 0.025 mm per pulse. When the pulses were accurately timed, movement was uniform and smooth. The deposition pattern of the LC effluent is shown in Figure 3. Deposition was accomplished in discrete steps linearly across the TLC surface. The steps are arranged in columns which run the length of the TLC plate. The linear coordinate along the path taken by the positioner could be transformed into a time coordinate. The transformation is performed knowing the number of steps taken and the delay time of the positioner a t each step during deposition. The positioner's rate of movement, data acquisition, and wavelength scanning were controlled by a microcomputer (2100, Zenith Data Systems, St. Joseph, MI) with software written in Turbo pascal version 111. The software allowed the operator to control such parameters as the delay time of deposition, the area of the TLC plate which was deposited with effluent, set-wavelength monitoring of the Raman band as a function of the position on the TLC plate, and the setting of the wavelength limits of the Raman spectrum as a function of the TLC position. The data were stored in computer memory and subjected to a nine-point Savitzky-Golay smoothing procedure ( 4 4 ) . The LC system consisted of a Shimadzu LC-6A chromatographic pump (Shimadzu Scientific Instrument Co., Inc., Columbia, MD), a Shimadzu SCL-GA system controller, and a Rheodyne Model 7125 injector (Cotati, CA) fitted with a 5-pL injection loop. Unless otherwise stated, the mobile phase consisted of 80% water and 20% methanol. The activated sol was pumped into the system with an ISCO syringe pump (ISCO, Lincoln, NE). In those experiments involving the mixing of the sol with the LC effluent, the mixing was accomplished with a low volume mixing tee.
RESULTS AND DISCUSSION The parameters that were optimized in order to maximize the signal intensity and preserve the LC peak shapes were (1) the time delays during deposition of the analyte to the TLC plate, (2) the total flow rate of the mobile phase and the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
Table I. FIA Peak Height and Width of 5 X 10” M Pararosaniline Measured as a Function of the Deposition Delay Time” delay, s/step 2.0 3.0 4.0 5.0
peak height: countsjs 4428 8200 5904 5822
(f227) (f525) (*332) (*305)
203GC,
,
,
,
1441
,
1
peak width: steps 33 21 14 12
a The laser power on the sample was 100 mW with the intensity monitored at the 1593-cm-’ band of the dye. *The standard deviations are given in parentheses. The peak widths were measured at the base of the peak.
postcolumn added sol, (3) the spacing between the “spots” of the deposited LC effluent and the distance between the rows of the deposited spots, and (4) the flow rate of the activated sol relative to that of the mobile phase. The flow rate of the sol and mobile phase in the LC/TLC system required that it be kept at a minimal value to prevent oversaturation of the aluminum oxide surface at any one depositon spot. Conventional LC flow rates of 1-2 mL/min were found to deliver a considerable excess of solvent onto the TLC surface. Thus, it was necessary to consider microbore or capillary LC systems operating at a flow rate of 10-200 pL/min. For a volumetric flow rate of 100 kL/min, for example, with a mobile phase composition of 80% water and 20% methanol, the diameter of the deposited spot was found to be ca. 5 mm for a deposition time of 3 s. The total volume of solution deposited was 5 pL/spot. Therefore, the spacing between the individual spots and the rows containing the successive spots must be greater than 5 mm in order to prevent overlap. When the Ag sols were deposited onto the TLC plate through the Teflon tube (flow rate and delay time identical with the mobile phase deposition), the diameter of the spots was ca. 1 mm. The decrease in the spot size is believed to be due to the strong interaction between the Ag particles and the hydrophilic aluminum oxide of the TLC plate. Also, the Ag particles may have a lower transport rate while increasing the total surface area for solvent adsorption. The consequence of the smaller spot size is %fold. First, the surface concentration of the test dye, pararosanaline, adsorbed on the Ag sol will be higher than the case when the dye is dispersed onto the TLC plate without the Ag sol. This localization of the dye will give a higher sensitivity for the SERRS analysis. Second, the distance between the deposition spots and rows can be reduced thereby reducing the area of the TLC required for a complete LC chromatographic run. The flow rate of the sol relative to the LC mobile phase and the deposition delay time were optimized by performing multiple injections of 1 X M pararosaniline while monitoring the signal intensity of the 1593-cm-I SERRS band of the dye. The optimal flow rates of the activated sol and the mobile phase were 70 and 30 pL/min, respectively. The decrease in the signal when the flow rate of the sol was much less than that of the mobile phase was probably due to the lack of a sufficient number of adsorption sites as the sol-dye ratio decreased. On the other hand, a decrease in the SERRS intensity was also found when the flow rate of the sol-mobile phase increased to more than the 70:30 ratio because of dilution to the analyte. The results of the peak intensity vs deposition time delay are given in Table I with the width of the resultant peak (as measured at the base). The peak width is essentially independent of the depositon time. The maximum signal intensity occurred when the deposition delay time was 3 s. Shorter deposition times cause the adsorbed dye to be deposited over a larger number of spots yielding a low surface concentration of dye per spot. When the deposition time is too long, the
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adsorbed dye spreads on the surface to a size larger than the probe area of the laser beam. Thus, all of the dye on the surface is not sampled and the SERRS intensity is less than what is expected. In Figure 4,the flow injection analysis (FIA) peak for an injection of 1 X M pararosaniline is shown. The peak width is very broad and non-Gaussian shaped. Subsequent injections resulted in further peak broadening. The broad and irregular shape of the peaks is a consequence of the small diameter deposition tube becoming blocked by the large aggregates which are apparently formed after the dye is adsorbed on the surface of the Ag sols. Although the resident time of the dye in the tube is only about 2 min for a volumetric flow rate of 100 pL in a tube with a length of 30 cm, the aggregation of the colloidal particles occurs rather rapidly upon addition of the dye. This aggregation can be observed by the change in the color of the sol with a corresponding shift in the absorption maximum in the transmission spectrum to longer wavelengths. In order to restore the signal intensity and the peak shape as seen in Figure 4, the deposition tube had to be washed with dilute nitric acid followed by water rinses. To eliminate the above problem, an alternative protocol for the addition of the sol to the dye was attempted. In this procedure, the deposition tube was connected directly to the injector (eliminate the mixing tee). The dye was then injected into the LC system and deposited directly onto the TLC plate. The solvent was allowed to evaporate and then the same path was retraced with the deposition tube now connected to the syringe pump for the addition of activated sol on top of the deposited dye. Figure 5 shows the FIA peak for the injection of 1 X low5M solution of pararosaniline. The volumetric flow rates for the dye and the sol depositions were 50 and 80 kL/min, respectively. The background corrected photon count was 9350 counts/s (f210 counts/$ In comparison, the peak intensity was 16 600 counts/s for the peak shown in Figure 4 when the mixing tee was used and the sol was mixed with the LC effluent containing pararosaniline. In an attempt to increase the peak intensity, the volumetric flow rate of the sol was increased while maintaining the LC flow rate and the deposition delay time (3 s/spot) constant. The results are tabulated in Table 11. As may be seen, the peak intensity of the 1593-cm-l band of the dye increased when the flow rate of the sol increased to a maximum at the flow rate of 160 pL/min. At faster flow rates, the intensity decreased. At the 160 pL/min flow rate with the 3 s/spot de-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
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Steps ( / 3 . O s e c ) Figure 6. Multiple injections of pararosaniline for the concentrations of 1 X 5X 1 X lo-', and 5 X lo-' M. The inset shows the calibration plot over this concentration range. The flow rate of the mobile phase was 50 pLlmin and the sol flow rate was 160 pL/min with a deposition delay time of 3 s. The laser power on the sample was 100 mW, and the 1593-cm-' band of the dye was monitored. Table 11. Peak Intensity of 1 X 10" M Pararosaniline as a Function of the Volumetric Flow Rate of the Sol Added to the TLC PlateD flow rate, pL/min
intensity,b
flow rate,
counts/s
pL/min
peak intensity,b counts/s
80.0 100.0 120.0 140.0
8625 (f135) 11125 (f179) 13 250 (*236) 15375 (*264)
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17625 (*290) 15000 (3~256) 11344 (f201)
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position time, 80 p L of sol was deposited per spot. Further increases in the flow rate of the sol resulted in an excess of the sol being deposited with the dye spreading on the TLC surface. The SERRS signal intensity decreased because of the decrease in the dye concentration within the interrogated area of the laser beam. Comparison of the peak intensities for the injection of 1 X M pararosaniline with simultaneous sol additon, accomplished by the mixing tee (see Figure 4), versus the second method of adding the sol onto the deposited dye indicated similar SERRS peak intensities. The favorable SERRS intensities demonstrated for the dye deposited onto the TLC plate followed by sol addition indicate that the dye shows preferential affinity for the sol surface. However, the peak shape and width were much improved in the latter situation. Continued addition of sol on the predeposited dye did not increase the peak intensity nor change the shape of the SERRS band. The observation of SERRS with the citrate-produced Ag sols suggests that efficient coupling of the incident electric field to the surface plasmons of the Ag surface is retained when the dye is predeposited on the TLC surface. This is a necessary condition of large enhancements (45). The transmission spectrum of the sols showed a broad absorption band. Scanning electron microscopy micrographs of the sols revealed that the broad absorption band was from the formation of linear aggregates in these citrate sols. Such aggregates are necessary in the activated sol, since the addition of the sol to the deposited dye limits the degree of aggregation.
750
'000
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'500
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R C I T ~ ~57 f l ( c T * * - I ' Figwe 7. SERRS spectrum of 1 X lo3 M pararosanilii acquired after the FIA of the dye. The spectrum was taken over the range of 375-1710 cm-' with a laser power of 100 mW on the sample. Flow rate of dye = 50 FL/min, flow rate of sol = 160 pL/min. Addition of the sol to the dye was accomplished on the TLC plate.
A calibration plot of the SERRS intensity vs pararosaniline concentration as obtained by FIA is shown in Figure 6. The dye was deposited onto the TLC plate with a delay time of 3 s and a flow rate of 50 pL/min, followed by the addition of the sol a t a flow rate of 160 pL/min. The SERRS intensity is linearly related to the dye concentration over the range of 1X to 3 X lo-' M. The correlation coefficient for the plot is 0.9998 (slope, 1645; intercept, 188 counts/s). The detection limit was 750 fmol (5 pL injection of a 1.5 X lo-' M dye) at a signal-to-noise ratio of 3. In addition to set-wavelength experiments, the SERRS spectrum was acquired as shown in Figure 7. The exact location of the dye on the TLC surface with the larger SERRS intensity was first determined by set-wavelength scanning of the surface. Knowing the location (x-y coordinates), the computer positions the "pen"holder carrying the optical fibers to this position for the acquisition of the SERRS spectrum. Since the LC flow is decoupled from the detection with the dye fixed on the TLC surface, the signal to noise for acquiring the spectrum can be improved by extending the photoncounting integration time. The well-defied SERRS spectrum shown in Figure 7 is for 1 x loT5M pararosaniline. The
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
'500
1600
1443
1700
R a r r a n sbift (cp?**--l) Figure 9. SERRS spectrum of 1 X lo-' M pararosaniline with 0% (a), 12.5% (b), and 25% (c)acetonbile added to the sol/dye mixture. The solldye mixture was added to the TLC plate with a microsyringe (20 pL). The spectra were taken over the range of 1400-1700 cm-' with a laser power of 100 mW on the sample.
Flgute 8. Three-dimensionalplot of time (TLC position) vs Raman shift vs intensity for the FIA of 1 X M pararosaniline. The spectrum was taken over the range of 1400-1700 cm-' with 100 mW of laser power on the sample. Flow rate of sol = 160 KL/min, flow rate of dye = 50 pL/min. The sol was added to the dye on the TLC surface.
spectrum herein matches the SERRS spectrum reported earlier (20). The SERRS spectra can also be determined as a function of the x-y coordinates of the TLC plate and plotted threedimensionally as Raman shift vs TLC position vs SERRS intensity (see Figure 8). The SERRS spectra of the deposited pararosaniline were acquired over the range of 1400-1700 cm-'. As may be seen from Figure 8, the SERRS bands at 1525 and 1593 cm-' begin to appear above the background when the positioner approaches the location on the TLC plate where the dye had been deposited. Also, interfering Raman bands from the TLC plate itself were not found over the 400- to 2000-cm-' range. This is a decisive advantage compared to Fourier transfer infrared spectroscopy in which broad and strong bands due to the TLC substrate are found and need to be subtracted from the analyte spectrum (40, 41). The acquisition of SERRS spectra, such as shown in Figure 8, can be helpful for identification purposes, particularly when LC bands are not totally resolved and contain impurities or overlapping analytes. One problem that can be envisioned when SERS is coupled to LC is a change in the adsorptive behavior of the analyte to the Ag surface in the presence of different types of LC organic mobile phases. Since SERS is a short-range effect, the maximum enhancement results from the chemisorption of the analyte molecules to the active adsorption sites of the Ag colloidal particles. The composition of the mobile phase, especially organic modifiers, may have a number of effects on the SERS spectral intensity. Some of these possible effects are the direct adsorption of the mobile phase to the active sites on the colloidal particle, the solubilization of the analyte thereby diminishing its affinity for the Ag surface, or affecting the aggregation behavior of the Ag colloid. In Figure 9, a portion of the SERRS spectrum for 1 X lo* M pararosaniline is shown as a function of added acetonitrile. The back-
ground-corrected intensity of the 1593-cm-' band was 15161 counts/s (f240 counts/s) with 0% acetonitrile and then 9222 counts/s (f131 counts/s) for 25% acetonitrile. As is evident from these spectra, acetonitrile has a detrimental effect on the SERRS intensity of pararosaniline. However, no Raman bands assignable to acetonitrile were observed in these experiments. Thus, the decrease in the SERRS intensity for pararosaniline may not be due to displacement of this analyte from the Ag surface by acetonitrile adsorption. The other suggested mechanisms may be operative. In the next series of experiments, pararosaniline (1 x M) was injected into the liquid chromatograph and deposited onto the TLC plate with a mobile phase consisting of 100% acetonitrile followed by another injection with a mobile phase of 100% water. Before the addition of the sol to the TLC plate, the mobile phase was allowed to evaporate for 1 h. The deposition path was retraced in each case with the addition of Ag sol a t a flow rate of 160 pL/min and a delay time of 3 s. For the water-deposited pararosaniline, the peak intensity a t 1593 cm-l was 16600 counts/s (f223 counts/s). The peak intensity for the acetonitrile was 16 557 counts/s ( f 2 2 1 counts/s). It is clear that if the mobile phase is removed prior to the addition of the Ag sol, only the dye remains on the surface and the SERRS signal can be obtained without solvent interference. Thus, it appears that this procedure will allow the SERRS analysis of LC analytes on the TLC to be independent of the LC mobile phase composition.
CONCLUSION SERRS can be coupled with LC/TLC for the acquisition of Raman spectral information and applied to LC analyte detection. This is feasible with a procedure where the Ag colloidal particles are postdeposited on the TLC surface. Such a procedure prevents the continued aggregation of the colloidal particles which can be detrimental to microbore LC systems. The time-invariant nature of the deposited analytes on the TLC surface allows time independent spectral interrogations. Thus, SERRS spectra can be obtained with only a modest investment in spectroscopic equipment. A SERRS detection limit of 750 fmol was demonstrated for deposited pararosaniline. The aluminum oxide surface of the TLC plate facilitated the adsorption of the dye to the Ag surface resulting in intense SERS signals. The removal of the LC solvent prior to addition of the Ag sols provides considerable flexibility in the choice
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Anal. Chem. 1990, 62, 1444-1451
of LC mobile phases without worry of detrimental interferences to the Raman analysis.
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RECEIVEDfor review January 10,1990. Accepted April 4,1990. The financial support of this work by the National Science Foundation is greatly appreciated. We gratefully acknowledge the funds provided by the Kansas University Endowment Association for the purchase of the Ar laser. S.A.S. thanks the Pittsburgh Conference for the Summer Fellowship administered by the American Chemical Society.
Nondestructive Analytical Procedure for Simultaneous Estimation of the Major Classes of Hydrocarbon Constituents of Finished Gasolines Jeffrey J. Kelly' and James B. Callis* Center for Process Analytical Chemistry, Department of Chemistry, BG-IO, University of Washington, Seattle, Washington 98195 Near-Infrared (NIR) spectroscopy In both the short-wavelength (700-1200 nm) region (SW-NIR) and the long-wavelength (1100-2500 nm) reglon (LW-NIR) Is used for the simultaneous estlmatlon of the major classes of hydrocarbon constltuents (allphatlcs, aromatics, and oleflnlcs) of flnlshed gasollnes. The test requlres 1-3 cm3 of gasollne, Is nondestructive, and takes only a few minutes to perform. Two multlvarlate calibratlon approaches have been employed for data reduction: stagewise multlllnear regresslon and partial least squares. Both methods yield comparable results. Success Is crltlcally dependent upon training sets of wellcharacterlzed samples for whlch both NIR spectra and constituent values by reference methods are avallable. Accordingly, the use of near-Infrared spectroscopy Is suggested as a rapid method for the estlmatlon of quality parameters of flnlshed gasollnes.
INTRODUCTION There are currently a number of ASTM-sanctioned tests for evaluating the quality of motor fuels, including octane
* Corresponding author.
Permanent address: D e p a r t m e n t of Chemistry, T h e Evergreen State College, Olympia, WA 98505.
numbers, API density, Reid vapor pressure, bromine number, and total aromatics, olefinics, and aliphatics. These tests are time-consuming, are tedious to carry out, require large quantities of material, and must be performed by well-trained technical personnel in central laboratories. Such conditions are not suitable for environmental or consumer protection monitoring or for on-line analysis, as the tests take too long to provide the rapid information feedback needed for decision making. Of these quality evaluations, the determination of hydrocarbon classes by the fluorescence indicator method, ASTM D 1319 (I),is the least precise. It depends strongly on operator discretion and the nature of the substrate material used for the determination. Norris and Rawdon (2) and Campbell et al. (3)have enumerated shortcomings of this method, pointing out, among other things, the long analysis time and the poor resolution of the method. Other methods, such as mass spectroscopy ( 4 ) , high-performance liquid chromatography (HPLC) (5-7), nuclear magnetic resonance (8),and supercritical fluid chromatography ( 2 , 3 ) ,have been recommended for the determination of hydrocarbon classes. Each of the above methods provides advantages over the fluorescence indicator methodin terms of increased accuracy at the expense of relatively high-cost instrumentation and generally long (20-60-min) analysis times. Thus, such methods are not well
0003-2700/90/0382-1444$02.50/00 1990 American Chemical Society
