Anal. Chem. 1087, 59, 523-525 6D
-
5 0 -
sa 3.0
-
2 0 1.0
-
523
effluent under pressurized flow, and bubbles were created only in the outlet line from the detector, where the pressure was almost at 1atm. Therefore, there was no instability of electric current during the experiment. Column efficiencies did not differ with or without an applied voltage in our preliminary experiments. Although we applied an electric voltage on the whole column in the present experiment, there are some alternative ways of applying voltage on the column. For example, it is possible to apply voltage to only a portion of the column. This method is the combination of high-performance liquid chromatography and electrochromatoeraDhv. In the absence of a pressurized flow, the present system is turned to an apparatus of zone electrophoresis, whose column is packed with a fine silica-based support used for high-performance liquid chromatography. The above method might be one way to solve the problem of column reproducibility in zone electrophoresis. We are currently working in this area. Registry No. CSI, 80641-41-4; uracil, 66-22-8. "
0
'
-I6
-12
-8
-4 APPLIED
0
+4 VOLTAGE
+8
112
+I6
I KVI
Flgure 3. Retention times vs. applied voltages. Solute A and B were uracil and CSI, respectively. Experimental condttions were the same as those in Figure 2, except for applied voltages.
were almost constant in the whole range of applied voltage. So u(mob) of uracil would be nearly zero at the present experimental condition. As applied voltages per unit column length are quite high, u(mob) of a solute having a valence electron, such as CSI, is almost the same value or of the same order of Ru. Therefore, it will become possible in electrochromatography that two solutes can be separated if there is a small difference in their mobilities, even though, the two solutes have identical retention times under conventional column conditions. So this method might be useful in the separation of proteins. In the absence of pressurized flow and with high voltage, bubbles were generated in the injector, column, and detector due to Joule heating and reactions a t terminals. But with pressurized flow, bubbles were observed only in a outlet line from the detector. The gas generated was dissolved in the
1
"
LITERATURE CITED (1) Chromatography, 3rd ed.; Heftman, E., Ed.; Van Norstrand: New York, 1975; Chapters 2 and IO. (2) Otsuka, S.; Listowsky, L. Anal. Blochem. 1980, 102, 419-422. (3) O'Farrell. P. H. Sclence 1985, 227, 1586-1589. (4) Krauss, J. S.; Jonah, M. H. Clh. Chem. (Winston-Salem, N . C . ) 1982, 28, 2000-2001. (5) Antrim, R. F.; Scherrer, R. A.; Yacynych, A. M. Anal. Chlm. Acta 1984, 164, 283-286.
Takao Tsuda Nagoya Institute of Technology Gokiso-cho, Showa-ku, Nagoya-shi 466, Japan RECE~VED for review May 30,1986. Accepted October 3,1986. Part of this work was presented a t 9th International Symposium on Column Liquid Chromatography, Edinburgh, July 1-5, 1985. This work was supported by Grand-in-aid for Cancer Research from the Ministry of Education, Science and Cuture (No. 61010038).
Thermal Lens Spectrophotometry Using a Tunable Infrared Laser Generated by a Stimulated Raman Effect Sir: Thermal lens spectrophotometry is one of the most sensitive analytical methods to detect very weak absorption ( I , 2). However, the conventional method using a visible laser can be applied only to analysis of the molecule with an absorption band in the visible region. Most inorganic and organic molecules such as ammonia and hydrocarbons have, unfortunately, no absorption band in the visible and ultraviolet regions. Therefore, infrared absorption spectrometry is essential for their determinations. Thermal lens spectrophotometry using an infrared laser has already been reported to be useful for sensitive determination of organic species. In our previous study we used a continuous wave COz laser for ultratrace analysis of hydrocarbons such as alcohols and benzene derivatives ( 3 , 4 ) . On the other hand Bialkowski et al. demonstrated ultrasensitive detection of hydrocarbons such as dichlorodifluoromethane, chlorotrifluoromethane, and ethanol by a high-power pulsed COPlaser (5-7).Harris et al. also reported the use of an infrared H e N e laser (3.39 pm) for determination of 2,2,4-trimethylpentane (8). The COz laser is line-tunable from 9 pm to 11pm, so that 0003-2700/87/0359-0523$01.50/0
the bar graph spectrum can be measured. It is quite useful for assignment of the molecular species (3),but a completely tunable infrared laser is urgently required for more reliable assignment of the molecules. Such an approach may provide us a new analytical tool for high-resolution spectrometry of trace sample species. In this study we construct a simple Raman cell for frequency conversion from visible dye laser emission to infrared radiation. We use this tunable infrared laser for recording a thermal lens spectrum of ammonia in the gaseous phase. We also discuss its potential advantage for its amlication to trace analvsis.
EXPERIMENTAL SECTION Raman Shifter. Figure 1shows the constructed Raman shifter consisting of only commercially available parts. Two stainless steel union-T are connected with a stainless steel tube (210 mm long, 1/4 in. diameter). Two quartz rods (11mm long, in. diameter) are tightly fastened as windows with Teflon ferrules in both sides. The end surfaces of the rods are polished and the cylindrical surfaces are ground to prevent the rods from slipping. The container is pressurized with hydrogen typically to 18 kg/cm2. 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
524
Laser Beam
I
k-
I
I
n
Stainless Tube
l
: 1
1
c
Stainless Tube
"1015 Quartz Window
1020
1025 1030 Wavelength (nm)
1035
1040
1
Flgwe 3. Thermal lens spectrum for ammonia in gaseous phase. Pure ammonia is used as a sample.
I H2 I
R o t a r y Pump
Figure 1. Raman shifter. Lena 2
B e e m Splitter I
F l o w Cell
Figure 2. Block diagram of thermal lens spectrophotometer using tunable infrared laser.
After being tested at 40 kg/cm2 for 1h in water, it was used for the experimental work. Exciting Source. We employed a dye laser (Lambda Physik, FL2002) pumped by a XeCl excimer laser (Lambda Physik, EMGlOBMSC). The laser dye was 7-(diethylamino)-4-methylcoumarin (Eastman Kodak Co., 7D4MC). It generated 4.2-mJ pulses in approximately 10 ns duration. For thermal lens spectrophotometry the third Stokes Raman emission (1015-1040 nm, 0.13 mJ) was utilized for the measurement of the spectrum. Other laser dyes of p-terphenyl (Dojindo Lab.) and rhodamine 6G (Daiwa Chemicals Factory) were also used for comparison. Arrangement of Optical System. Figure 2 shows a block diagram of the thermal lens spectrophotometer based on a dual beam system. The dye laser is focused into the Raman shifter by a quartz lens (Shigumakoki Co., focal length 500 mm). The induced Stokes and anti-Stokes beams are collimated by a quartz lens (ShigumakokiCo., focal length 200 mm), and the third Stokes beam is isolated by a color filter (Toshiba, R-63). It is focused into a 1-cm sample cell by a quartz lens (Shigumakoki Co., focal length 300 mm). A He-Ne laser (Uniphase, Model 1103) is used as a probe beam. It is coaxially aligned with the exciting beam by a wedged quartz plate, pawing through the sample cell without focusing the beam. The probe beam is isolated by a quartz prism and a band-pass filter (Ditric, 632.8 nm). The intensity at the beam center is measured by a photodiode (active area, 1 mm square). The waveform of the signal is measured by a transient digitizer (Autnics, S210,l kword memory) and is averaged by a signal averager (Autnics, F601). The system is controlled by a microcomputer through a GP-IB interface. The thermal lens spectrum is measured by a boxcar integrator (NF Circuit Design Block, BX530A) and displayed by a chart recorder (Rikadenki, R-50). Reagents and Procedure. The sample of ammonia and the nitrogen used for dilution were supplied from Iwatani. The
concentration of ammonia was adjusted by changing the mixing ratio of these gases. RESULTS AND DISCUSSION Stimulated Raman Effect. The maximum pulse energy was obtained for Stokes emission when the 7D4MC dye laser was focused by the lens with a 400-mm focal length. The typical pulse energies for first, second, and third Stokes emission were S1 = 0.29 mJ, S2 = 0.19 mJ, S3 = 0.058 mJ, respectively. On the other hand the maximum energies for the anti-Stokes emission were obtained when the laser was focused by the lens with a 250-mm focal length. The energies were AS1 = 0.059 mJ, AS2 = 0.013 mJ, and AS3 = 0.003 mJ, respectively. We could generate no stimulated emission for the rhodamine 6G laser (580 nm), but frequency conversion was more efficient for the p-terphenyl laser (340 nm). The present results are logical, since the gain for frequency conversion is proportional to the frequency of Raman emission (9). In this study we used third Stokes emission for excitation of ammonia. Thermal Lens Spectrophotometry. Figure 3 shows the thermal lens spectrum for ammonia in the gaseous phase. This spectrum consists of third overtone vibration (9760.4 cm-') of v1 band (3335.9 cm-', 3337.5 cm-') for ammonia (10). Many lines are originated by the rotational transitions. It is noticed that the peaks are slightly broadened, which seem to be due to pressure broadening since the measurement is carried out at an atmospheric pressure. The signal-to-noise ratio for the prominent line at 1025.69 nm was 30, at present. The straight analytical curve was constructed by diluting ammonia with nitrogen. The detection limit achieved by this line was 6% at a laser pulse energy of 0.13 mJ. The enhancement factor, the relative sensitivity in comparison with conventional absorption spectrometry, was experimentally determined to be 2.5 from the slope of the analytical curve. On the other hand the theoretical value was 25, which was calculated from the aberrant lens model (11). The observed value is apparently smaller than the theoretical one. Discrepancy might be originating from poor beam quality of the dye and stimulated Raman laser. It is noticed that rather poor detection limit is coming from a small molar absorptivity (e = 0.05) for ammonia and from a small enhancement factor. At present, the sensitivity of this method is limited by the output power of the exciting source. The state-of-the-art Nd:YAG-laser-pumped dye laser produces 150-mJ pulses at 560 nm, which is converted to 14-mJ pulses at 1048 nm by a Raman shifter (12). A more recent publication informs that the conversion efficiency can be improved to 80% by using a Raman laser amplifier system (8). Then, the detection limit can readily be improved more than 2 orders of magnitude by using such an exciting source. It is noteworthy that the resolution of the spectrum can be improved by reducing the
525
Anal. Chem. 1987, 59, 525-527
total preasure of the sample and the line width of the dye laser The sensitivity be for further of pulsed thermal lens spectrophotometry is reported to be pressure Of the independent to the (I3)* Therefore, we expect that the present method is very promising for high-resolution spectrometry of trace molecules in the infrared region. Registry No. NH3, 7664-41-7.
(9) Hanna. D. C.; Pacheco, M. T. T.; Wong, K. H. Opt. Commun. 1985. 55, 188-192. (10) Herzberg, G. Molecular Spectra and Molecular Structure I I Infrared and Raman Spectra of Po&atomic Molecules; Van Nostrand Reinhokl: New York, 1945; pp 294-297. (11) Sheldon, J. S.; Knight, V. L.; Thorne, M. J. Appl. Opt. 1982, 27, 1663-1 669. (12) Specification for Nd:YAG laser (YG581C) pumped dye laser (TDLBO), Ouantel International, Santa Clara, CA, June, 1986. (13) Mori, K.; Irnasaka, T.; Ishibashi, N. Anal. Chem. 1983, 5 5 , 1075- 1079. ~
Shuichi Kawasaki Totaro Imasaka Nobuhiko Ishibashi*
LITERATURE CITED (1) Harrls, J. M.; Dovichi, N. J. Anal. Chem. 1980, 52, 695A-706A. (2) Imasaka. T.; Ishibashi, N. Trends Anal. Chem. 1982, 7 , 273-277. (3) Hlgashl. T.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1984, 56, 2010-2013. (4) Hlgashi, T.; Imasaka, T.; Ishibashi, N. Bunseki Kagaku 1982, 31, 680-68 1. ( 5 ) Long, G. R.; Bialkowski, S. E. Anal. Chem. 1984, 56, 2808-2811. (6) Nickolaisen, S. L.; Bialkowski, S. E. Ana. Chem. 1985. 57, 758-762. (7) Long. G. R.; Blaikowskl, S. E. Anal. Chem. 1986, 58, 80-86. (8) Carter, C. A.; Brady, J. M.;Harris, J. M. Appl. Speclrosc. 1982, 36. 309-314.
Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812, Japan RECEIVEDfor review August 5, 1986. Accepted October 1, 1986.
Direct Analysis of High-Performance Thin-Layer Chromatography Spots of Nucleic Purine Derivatives by Surface-Enhanced Raman Scattering Spectrometry Sir: Direct photometric identification of high-performance thin-layer chromatography (HPTLC) spots is usually achieved by plotting absorption spectra in reflectance and fluorescence spectra ( I ) . Indeed methods for vibrational spectra such as infrared and Raman spectrometries are limited by the required high quantities of substances and spectral properties of the adsorbent (2,3). However, recent findings of an enhanced Raman scattering for various compounds adsorbed at the silver surface (SERS, surface-enhanced Raman scattering) (4,5) give new potential for analytical applications of Raman spectrometry (6). Thus spraying paper chromatograms with silver colloidal solution produces a strong intensity for resonance Raman scatterers from separated dyes (7,8).This surfaceenhanced resonance Raman scattering (SERRS)spectrometry has permitted in situ colored substances down to 2 ng/cm2 to be detected and identified from chromatogram spots (7). However, investigations with colorless sustances (nonresonance Raman scatterers) in silver colloidal solution have also shown strong enhancement of vibration intensity far from resonance conditions (4,5). Thus purine derivatives from nucleic acids can be detected a t concentrations as low as 10" M and identified by the fingerprint region of their spectra (below 2000 Furthermore application of minute volumes of cm-I) (9,10). silver colloidal solutions with guanine derivatives on HPTLC silica gel 60 support enables SERS spectral recordings to be carried out at subnanogram levels (11). In this paper a method has been developed to record in situ HPTLC chromatogram spots of alkylated nucleic purine derivatives. The presence and the variable extent of such modified nucleic bases in biological extracts have been correlated to tumorigenicity (12, 13).
EXPERIMENTAL SECTION Guanine (Gua), adenine (Ade), and purine were obtained from Serva, Heidelberg (FRG). 1-Methylguanine (1-MeGua), 3methylguanine (3-MeGua), 7-methylguanine (7-MeGua), and 9-methylguanine (SMeGua) were purchased from Fluka,Neu-Ulm (FRG). 1-Methyladenine (1-MeAde) and 1-methybypoxanthine (1-MeHyp) were purchased from Sigma (St. Louis, MO). OeMethylguanine (OB-MeGua) was prepared in our laboratory (10). All other chemical reagents were of analytical quality and were
purchased from E. Merck (Darmstadt, FRG). HPTLC plates of silica gel 60 (10 cm X 10 cm) without fluorescent indicator are produced by E. Merck. The silver colloids were prepared by use of Creighton's procedure (5) by the reduction of AgN03 with N&Hk In a typical experiment, one volume part of M AgN03 is added dropwise to three volume parts of 2 X M NaBH,, cooled in an ice bath, and mixed vigorously. The yellow brownish silver colloidal solution was stored at 5 "C for weeks without any change in color. For HPTLC chromatography, single-compound stock solutions of nucleic purine derivates were prepared to yield a concentration of about M in ethanol/water (1/1)or methanol (guanine 5 X lo4 M). Application of volumes of solution down to 100 nL has been undertaken with a 1-pL Hamilton syringe in conjunction with a micrometer. The nucleic purine derivative separation was accomplished by using a mixture of chloroform, methanol, and ammonia (60:201) (14). Development time for ascending chromatograms takes about 15 min in a covered glass tank. After drying, HPTLC plates are sprayed to wetness with silver colloidal solution by a spray atomizer. Colored spots arising on the HPTLC plate are used to locate the separated nucleic purine derivatives. The spot color varies from pale yellow through orange to violet depending on the nature and concentration of compounds. As already observed in solution (9-11) aggregated silver colloids induced by adsorption of purine derivatives are responsible for the colored spots. HPTLC plates are analyzed at room temperature by a computer-controlled double beam spectrometer: Spex double monochromator 14018 (0.85 m, f/7.8), Datamate DM 1, cold photomultiplier (RCA 31034 A) operated in the photon counting mode. Monochromator slits were selected so as to provide better than 8 cm-* band-pass. The excitation wavelength was the 514.5-nm line of an argon ion laser (Spectra Physics, Model 2020-03) with 10 mW of power. HPTLC-SERS spectra were measured in a typical 90"scatteringarrangement. Spot diameters are about 2-4 mm and the laser beam focused on HPTLC plates is approximately 1.5 mm in length and 0.1 mm in width. Spectra were obtained with a scanning speed (accumulation time) of 2 cm-'/1.5 s for ratios of substance quantity/spot from 10 to 60 ng. RESULTS AND DISCUSSION Previous observations have led to the conclusion that normal Raman spectrometry is not a very promising technique for in situ analysis of TLC spots (3). Indeed very high con0 1987 American Chemical Society
