Anal. Chem. 1991, 63,437-442
per molecule per transit time), one obtains
LIYeyU
The value taken for g,, at T = 0 is that arrived a t by extrapolating the autocorrelation function at T > 0 to T = 0 (see Figure 6) and subtracting the average background level (g,). The values for S and b are taken as the average values. As can be seen from eq 8A, the number of photons per molecule per transit time can be calculated from the magnitude of the autocorrelation function at the extrapolated T = 0 value when
S < b.
LITERATURE CITED (1) Saunders, G. C.; Jett, J. H.; Martin, J. C. Clin. Chem. 1985, 31. 2020. (2) Jett. J. H.; Keller, R. A.; Martin, J. C.; Marrone, B. L.; Moyzis, R. K.; Ratliff, R. L.; Seitzinger, N. K.; Shera, E. B.; Stewart, C. C. J . Biomol. Struct. Dyn. 1 9 8 9 , 7 , 301. (3) Davis. L. M.; Fairfield. F. R.; Jett, J. H.; Keller, R. A,; Hahn, J. H.; Krakowski, L. A,; Marrone. B. L.; Martin, J. C.; Ratliff. R. L.; Shera, E. B.; Soper, S. A. Genet. Anal.. in press. (4) Dovichi, N. J.; Martin, J. C.: Jett. J. H.: Trkuia. M.: Kelier. R. A. Anal. Chem. 1984, 56, 348.
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(5) Dovichi, N. J.; Martin, J. C.; Jett, J. H.; Keller. R. A. Science 1983, 219, 845. (6) Nguyen, D. C.; Keller, R. A.; Trkula, M. J. Opt. Soc. Am. 1987, 4 , 138. (7) Jett, J. H.; Keller, R. A.; Martin, J. C.; Nguyen, D. C.; Saunders, G. C. Flow Cytometry and Sorting; Wiley-Liss: New York, 1990; pp 381-396. (8)Hahn, J. H.; Soper S. A.; Nutter, H. L.; Martin, J. C.; Jett, J. H.; Kelier, R. A. Appl. Spectrosc., in press. (9) Nguyen, D. C.; Keller, R. A.; Jett, J. H.; Martin, J. C. Anal. Chem. 1987, 59, 2158. (10) Peck, K.; Stryer, L.; Glazer, A. N.; Mathies, R. A. Roc. Natl. Acad. Sci. U . S . A . 1989, 86, 4087. ( 1 1) Shera, E. B.; Seitzinger, N. K.; Davis, L.; Keller, R. A,; Soper, S. A. Chem. Phys. Len. 1990, 174, 553. (12) Mathies, R. A.; Peck, K.; Stryer, L. Anal. Chem. 1990, 62, 1786. (13) Soper, S. A.; Davis, L. M.; Jett, J. H.; Martin, J. C.; Nutter, H. L.; Shera, E. B.; Keller, R. A. Manuscript in preparation. (14) Alfano, R. R.; Shapiro, S. L.; Yu, W. Opt. Commun. 1973, 7 , 191. (15) Kubin, R. F.; Fletcher, A. N. J. Lumin. 1982, 2 7 , 455. (16) Skogen-Hagenson, M. J.; Salzman, G. C.; Mulianey, P. F.; Brockman, W. H. J. Histochem. Cytochem. 1977, 25, 784. (17) Watson, J. V. Br. J. Cancer 1985, 51, 433. (18) Watson, J. V. Cytometry 1989, 10, 681.
RECEIVEDfor review September 11,1990. Accepted December 13, 1990.
Determination of Purine Bases by Reversed-Phase High-Performance Liquid Chromatography Using Real-Time Surface-Enhanced Raman Spectroscopy Rongsheng Sheng
Center of Analysis and Measurement, W u h a n University, Wuhan, Hubei, People's Republic of China Fan Ni
Diagnostics Laboratory of Veterinary College, Iowa State University, Ames, Iowa 50011 Therese M. Cotton*
Department of Chemistry, Iowa State University, Ames, Iowa 50011
The determination of four purine bases (adenine, guanine, hypoxanthine, and xanthine) by reversed-phase high-performance liquid chromatography (RP-HPLC), in combination with real-time surface-enhanced Raman spectroscopy (SERS) detection, is demonstrated. The goal of this study was to examine several factors (laser irradiation, pH, memory effects, and the construction of the interface between the RPHPLC system and the Raman spectrometer) that affect SERS detection under flowing conditions. The separation and detection of a mixture of four purine bases was accomplished. The quantity of bases used for the analysis was 1 mmoi for adenine and guanine, 5 nmoi for xanthine, and 10 nmoi for hypoxanthine. Three-dimensional (SERS intensity, Raman shift, and chromatographic retention time) and two-dimensional (SERS intensity and chromatographic retention time) chromatograms are presented.
INTRODUCTION The detection and quantitation of nucleotides, nucleosides, and their bases has become increasingly important in the field
* Author to whom correspondence should be addressed.
of biomedical research. The presence of nucleic acid components in physiological fluids, tissues, and cells results from catabolism of nucleic acid, enzymatic degradation of tissues, dietary habits, and various salvage pathways. Changes in the concentration of these components may reflect substantial alterations in the activity of catabolic, anabolic, and interconversion enzymes and may be used to indicate the presence of various disease states which cause alterations in the normal purine and pyrimidine metabolic pathways. At present, high-performance liquid chromatography (HPLC) is considered the most promising method for the determination of purine and pyrimidine metabolites. It is also a powerful technique for monitoring the therapeutic response to purineand pyrimidine-based drugs and for determination of their effectiveness and toxicity (1-3). Although analysis of nucleic acid components can now be achieved rapidly, accurately, and with high sensitivity by HPLC, the assignment of individual peaks in highly populated chromatograms remains a major problem. Retention times are not sufficient for positive identification of separated components, unless additional information is available. U1traviolet (UV) spectroscopy has been widely used for this purpose, but the spectra are often difficult to assign unambiguously, especially when closely related or difficult to sep-
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arate compounds are considered ( 4 ) . Other techniques that are more suitable for the structural identification of analytes following HPLC separation, including mass spectrometry (MS) ( 5 ) , nuclear magnetic resonance (NMR) (6), and infrared spectroscopy (IR) (7),are currently under development. Each of these techniques has limitations, however, and improvements in methodology are continually under investigation. Raman spectroscopy (RS) provides structural identification of samples and has considerable potential for HPLC detection. One of the major advantages of Raman spectroscopy for HPLC detection, as compared to infrared spectroscopy, is the ability to examine biological samples in an aqueous environment. However, this advantage is offset by the inherently lower sensitivity of Raman scattering as compared to IR absorption. Resonance Raman scattering (RRS), surface-enhanced Raman scattering (SERS), and surface-enhanced resonance Raman scattering (SERRS) spectroscopies exhibit considerably greater sensitivity, and these variants of Raman spectroscopy are promising for hyphenated HPLC-Raman analysis. SERS has been used to identify nucleic acid bases and several of their derivatives (8-20). The emphasis of many of the studies has been on characterizing the SERS spectra of individual bases or their derivatives. The results have provided spectroscopic data and also identified the interrelationships between the SERS spectra and analyte concentration, pH, orientation of the adsorbate a t the surface, and conditions for SERS activity (such as roughness in the case of films and electrodes, applied potential in the case of electrodes, and particle size and aggregation state in the case of sols). These previous studies have provided support for the further development of SERS-based analysis of nucleic acid bases and their derivatives. Recently, the use of SERS as an analytical method for identifying nucleic acid bases and their derivatives has been explored. Koglin et al. (9) reported the SERS spectrum of a mixture containing adenine, guanine, cytosine, and thymine. The vibrational bands originating from the adenine base were dominant, and bands from other bases were relatively weak. Because of the spectral overlap, it is difficult to distinguish the four bases clearly in the spectrum. Carrabba et al. (21) and Laserna et al. (22) have also used this approach with organic compounds. Again, because no prior separation step was used in these studies, it is difficult to identify some of the individual components in the spectra. The previous results indicate that a prior separation step is desirable for SERS analysis of mixtures. Analytical applications of SERS utilizing the high inherent structural sensitivity in combination with the excellent separation capability of chromatography have appeared in recent years (19,23-26). Tran (23) used SERS to identify three dyes separated by paper chromatography (PC). Sequaris and Koglin (19) used SERS to detect purine and nine of its derivatives following separation by high-performance thin-layer chromatography (HPTLC). Detection limits for individual compounds were estimated at 5 ng/per spot when the sample was excited with 10 mW of 514.5-nm light from an Ar+ laser. A similar study has also been conducted by Xiong and Sheng (2.11, who used SERS to detect adenine, guanine, cytosine, uracil, thymine, and several dyes separated by TLC or PC. Detection limits were in the nanogram to picogram range. Combined HPLC-SERS analysis is technically more difficult than the above techniques because the analyte fractions are flowing and conditions are continuously changing within the detection cell. However, because of the importance of HPLC in chemistry and biology, this approach has considerable appeal. Freeman et al. (25)were the first to investigate a SERS-based HPLC detection scheme. They successfully
interfaced an HPLC system with a Raman spectrometer and used colloidal silver as the SERS-active substrate. A detection limit of 100 ppb was achieved for a common organic dye, pararosaniline hydrochloride, as the test analyte. SERS spectra were obtained under stopped-flow conditions. This study demonstrates the considerable potential of combined HPLC-SERS, although only a single compound was tested rather than a mixture. Ni et al. (26) used SERRS as an off-line, ancillary HPLC detector for a mixture of nitrophenol compounds. Detection limits were near 14 ppb, and the SERRS spectra were recorded under static conditions. Thus, several attempts have been made to combine SERS with HPLC, but prior to now, most SERS measurements have been recorded under static conditions. Obviously, there are additional problems associated with utilizing SERS detection under flowing conditions. The postcolumn conditions of HPLC can be simulated by a flow injection analysis (FIA) system. Several researchers have interfaced SERS as a real-time detector with a FIA system. Under continuous-flowconditions, the major difficulty encountered is the maintenance of an active and clean SERS substrate, especially when several compounds are introduced sequentially into the system. By the use of a short electrochemical roughening procedure, For& was able to regenerate a SERS active-Ag electrode surface under flowing conditions and record SERS spectra of pyridine (27,28) and DNA bases (28). However, this method requires a reproducible roughening procedure, as well as a potential step to remove the adsorbate. The use of a Ag sol as a SERS substrate has several obvious advantages in a continuous-flow system, including the mobility of the Ag particles and the availability of a fresh silver surface for each of the separated analytes. Winefordner et al. (29-31) used SERS to detect p-aminobenzoic acid (PABA) and 9-aminoacridine (AA) by mixing these compounds with a Ag sol and passing the mixture through a FIA system. The effects of the Ag preparation procedure, concentration of reagents, sol activation time, interaction time for the aggregated Ag particles and the analyte, and pH of the flowing stream on SERS intensities were investigated. The spectral reproducibility was 3.2%, and the detection limit was 30 ng. The results of these investigations provide motivation for the development of a combined HPLC-SERS procedure. As a preliminary study to the combined HPLC-SERS analysis of bases, we have employed a FIA-SERS system to investigate the real-time analysis of four RNA bases injected sequentially into the system (32). The effects of several parameters on the SERS intensities of the bases under flowing conditions were demonstrated. These included the temperature of the eluant/Ag sol mixture, the pH and flow rate of mobile phase, and the tubing material used in the construction of the FIA system. Increasing the temperature of the analyte/Ag sol mixture was found to be a very effective method for increasing the SERS signal under flowing conditions. The present study pursues the goal of obtaining real-time SERS spectra of four purine bases following reversed-phase highperformance liquid chromatography (RP-HPLC) separation. Several parameters and the design of the interface between the RP-HPLC system and the Raman spectrometer were investigated. By carefully controlling the experimental conditions, the SERS spectra of the four purine bases were detected in real time. EXPERIMENTAL SECTION Reagents. All chemicals, including adenine, guanine, hypoxanthine, xanthine, sodium citrate, silver nitrate, and potassium dihydrogen phosphate (AR grade), were purchased from Sigma Chemical Company (St. Louis, MO 63178) and were used without further purification. The water was deionized and twice-distilled. Silver Sol Preparation. Silver sols were prepared by the M AgN03 citrate reduction procedure (33). One liter of a 1 X
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Figure 1. Schematic diagram of the HPLC-SERS system: (1) HPLC unit; (2) chromatographic column; (3) silver sol; (4)delivery tube; (5) peristaltic pump; (6) coiled tube for heating sol; (7) heating tape; (8) variable transformer; (9) thermometer; (10) 30-30 tee joint; (11) laser beam; ('t2) Raman cell; (13) waste solution; (14) collection lens; (15) entrance slit to monochromator; (16) monochromator; (17) intensified photodiode array detector.
20000
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moo
solution was degassed by heating to the boiling point. Twenty M sodium citrate solution was added milliliters of a 3.4 X dropwise to the AgNORsolution while stirring vigorously. The mixture was maintained at the boiling point for 1h, and the final volume was adjusted to 1000 mL with distilled water. The silver sol was transferred to a graduated cylinder, covered, and allowed to fractionate by sedimentation for 1 week. Successive 100-mL aliquots were carefully withdrawn from the cylinder for SERS analysis. The fraction exhibiting the maximum SERS enhancement was used as the substrate. Purine Base Stock Solution. Solutions of adenine and M), xanthine (2 X M), and hypoxanthine guanine (1 x M) were prepared in distilled water. The mixed base (4 X solution was prepared by combining equivolumes of each of the stock solutions. Chromatography. The chromatograph consisted of a Model 2350 ISCO HPIX pump, a Model 2360 ISCO gradient programmer, and a Model IV ISCO variable-wavelength UV/vis detector (ISCO, Inc., Lincoln, NE 68505). The RP-HPLC column was a NOVAPAK C18 column (15 cm X 3.9 mm i.d.) (Phenomenex, Torrance, CA 90501). Prior to use, all solutions and solvents were vigorously degassed and particulate matter was removed by passage through a 0.45-pm filter under vacuum. The HPLC separations were preformed by isocratic elution using 0.01 M potassium dihydrogen phosphate solution at pH 4.5 as the mobile phase. The flow rate was 0.30 mL/min. The sample injection volume was 10 pL. Analyte elution was monitored at 254 nm (with the UV detector) and at 1462 and 1730 cm-' (with the SERS detector). Raman Instrumentation. the 476-nm line of a Coherent Innova 100 Kr+ laser was used as the excitation source for Raman scattering. The laser power was 40 mW at the sample. The excitation beam was at 45" relative to the surface normal, and the Raman scattered light was collected in a backscattering geometry. The light was dispersed by a Spex Triplemate spectrometer and detected by an intensified silicon photodiode array detector (Model 1421-R-1024HD, Princeton Applied Research Corp.) which was cooled to -40 "C. The spectra were recorded and processed with an optical multichannel analyzer (OMA-3, Model 1460, Princeton Applied Research Corp.). Two spectra were summed by using the data acquisition mode 6 and a 10-s exposure time per scan. The 92 spectra obtained in a single chromatographic run were stored in sequential memories. Coupled HPLC-SERS System. A schematic diagram of the coupled HPLC-SERS system is shown in Figure 1. It includes the following (numbers in parentheses refer to those in the figure): (1,2) HPLC system for separating the purine bases and pumping them to the tee joint; (5) a peristaltic pump (Buchler Instruments, Fort Lee, N..J.) to pump the Ag sol solution through the heated, coiled tubing and to the tee joint; (6) the coiled tubing (stainless steel, i.d. 0.5 mm) for heating the Ag sol to above 90 "C; (10) tee joint for mixing the separated bases with the heated Ag sol (the angle between the entrance and outlet tubing is 30"); (11)Raman flow cell consisting of a glass capillary (100 X 1.0 mm i.d.) fitted into an aluminum holder; (16) Raman spectrometer and OMA-3.
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Figure 2. SERS spectra of the four purine bases obtained under static conditions and at room temperature. The pH of the solutions was 4.5, and the concentrations of the bases were as follows: A and G = 2 X M; X = 1 X H=2 X M. The laser power at the sample was 40 mW. The spectral acquisition time was 25 s/scan.
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IZ
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Figure 3. SERS intensity of adenine as a function of irradiation time. The solution pH was 6.0, and the adenine concentration was 5 X M. Spectra were recorded at room temperature and under static conditions. The laser power was 40 mW, and the spectral acquisition time was 20 s.
RESULTS AND DISCUSSION The SERS spectra of four purine bases (adenine (A), guanine (G), xanthine (X), and hypoxanthine (H)) obtained a t room temperature and under static condition are shown in Figure 2. Bands corresponding to the ring breathing modes for A, G, X, and H are a t 732, 652, 658, and 722 cm-l, respectively. In addition to the ring breathing modes, bands within the higher wavenumber region are also quite different in the spectra of the four bases. It can be seen that each base is readily distinguished from the others by the band positions and relative intensity patterns. Thus, the SERS spectra provide a distinctive fingerprint for each purine base. It was determined in a previous study (32) that SERS spectra of nucleic acid bases were quite strong under static conditions but were below the detection limit or very weak under flowing conditions. The decrease in signal intensity is a result of the short irradiation time experienced by a flowing sample in the laser beam. Figure 3 clearly shows that the adenine SERS spectrum cannot be observed during the first of a series of scans under static conditions. The SERS spectrum is detectable after tens of seconds of irradiation, and the intensity increases to a maximum after approximately 6 min of irradiation. The irradiation time required for the appearance of the maximal SERS spectrum or the "critical irradiation time" is dependent upon the identity and concentration of the analyte and the pH and temperature of the
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Table I. Some Physicochemical Properties of Purine Bases Durine bases H G
A pKb
