Anal. Chem. l907, 59, 521-523
coustic detector; however, since sound is generated only when the laser frequency coincides with a molecular absorption, it would appear that the selectivity of this detection should be one of its salient features. In fact, previous workers have noted this property of optoacoustic detectors and have made quantitative estimates of their capability for trace detection in the presence of other gases (3). Although the optoacoustic detection technique described here is general and can be applied, in principle, to any species that possess an infrared absorption, the compounds studied here (with the exception of SF6)have been chosen on the basis of their relevance to topical environmental contamination problems. Ethylene oxide is widely used as a sterilizing agent and is a feedstock for the production of a number of industrial chemicals. It is known to be mutagenic (23)and carcinogenic (24,25). A derivatization technique has been developed (26) for use with gas chromatography that gives a detection limit of 56 ng in a flame ionization detector. PCB’s have been used in transformer oils and hydraulic fluids, and as paint additives, dielectric materials, and plasticizers. They have been found as accidental contaminants in air, water, sediment, and food samples (27,281. PCB’s have acute toxic effects (28,29)and have been found to cause tumors in laboratory animals (30-32). Various methods for trace analysis of PCB’s in environmental samples have recently been reviewed (33, 34); electron capture detection of PCB’s has been reported with a sensitivity of 0.5 ng (34). Vinyl chloride, used extensively in the United States for the production of poly(viny1chloride), has been documented as mutagenic and carcinogenic (35). The detection limit (36) for vinyl chloride using electron impact ionization mass spectrometry with gas chromatography is 0.1 ng. Polychloronaphthalenes represent a lesser public health hazard than the above species; their toxic and carcinogenic properties have been reviewed in ref 37 and 38. The detection limit (36) for 1-chloronaphthalene using single ion mass spectrometricmonitoring of the output of a gas chromatograph is 0.15 ng. SF6 is generally considered to be nontoxic. It can be detected to 0.5 fg with an electron capture detector (36). The work done here with a gas chromatograph serves to point up the contrast between trace detection of concentrations (measured in, for instance, part per billion) and quantities (measured in grams) of a given species. In the former case (e.g., monitoring atmospheric NO concentrations), there are, in general, no restrictions on the sampling volume. As is well-known, the sensitivity of optoacoustic detection is limited by the small signal that emanates from the cell windows. (Note that a few techniques (39-42) have been investigated to reduce this problem.) In the case of trace detection to determine concentrations of analytes, it is possible to use a long cylindrical gas volume to reduce the acoustic energy per unit volume of the window signal without a sacrifice in the acoustic signal amplitude from the analyte. On the other hand, when the sample volume is restricted, as in the case of a detector for gas chromatography, the problem of absorption at the cell windows becomes acute. Despite this limitation, the minimum detectable concentration reported here for the small volume cell is comparable with that previously reported for SF6and a number of other gases (3,43); such sensitivity attained with the use of a relatively
521
straightforward cell design and conventionalsignal processing techniques gives testimony to the remarkable capabilities of the optoacoustic effect itself.
ACKNOWLEDGMENT The authors are grateful for the support of this research by the National Institutes of Health. LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
(16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34)
Kerr, E. L.; Atwood, J. G. Appi. Opt. 1968, 7 , 915. Kreuzer, L. B. J. Appi. fhys. 1971, 4 2 , 2934. Kreuzer, L. B.; Kenyon, N.; Patel, C. K. N. Science 1072, 777, 347. Goldan, P.; Goto, K. J. Appl. fhys. 1074, 45, 4350. Kreuzer, L. B. Anal. Chem. 1978, 5 0 , 597A. Zharov, V. P.; Montanari, S. G.; Tumanova, L. M. Zh. Anal. Khim. 1084, 39,551. Choi, J. G.; Diebold, G. J. Anal. Chem. 1985, 5 7 , 2989. Monahan, E. M.,Jr.; Nolle, A. W. J. Appi. fhys. 1977, 48, 3519. Quimby, R. S.; Selzer, P. M.; Yen, W. M. Appl. Opt. 1077, 16, 2630. Murphy, J. C.; Aamodt, L. C. J. Appl. fhys. 1077, 4 8 , 3502. Fernellus, N. C. Appi. Opt. 1979, 78, 1784. Busse, G.; Herboeck, D. Appi. Opt. 1979, 18, 3959. Shaw, R. W. Appl. fhys. Lett. 1979, 35, 253. Bechthold. P. S.;Campagna, M.; Schober, T. Solid State Commun. 1080. 36. 225. Bechthold, -P. S.; Campagna, M.; Chatzipetros, J. Opt. Commun. 1981. 36. 369. Bechthold, P. S.;Campagna, M. Opt. Commun. 1981, 3 6 , 373. Morse, P. Vibration and Sound; McGraw-Hill: New York, 1936. Pelzl, J. Appl. fhys. 1981, 2 5 , 221. Nordhaus, 0.; Pelzl, J.; Klein, K.; Nordhaus, 0. Appl. Opt. 1082, 2 7 , 94. Lagemont, R. T.; Jones, E. A. J. Chem. fhys. 1051, 79, 534. Hummel, D. 0.; Scholl, F. Infrared Analysis of Polymers, Resins and AWitives: An Atlas; Why-Interscience: New York, 1969; Vol. I . Choi, J. G. Ph.D. Thesis, Brown University, 1985. Ehrenberg, L. Banbury Rep. 1979, 7 , 157. Dunkelberg, H. Br. J. Cancer 1979, 39, 588. Coene, R. F. Vet. Hum. Toxicol. 1981, 2 3 , 439. Nagase, M.; Kondo, H.; Mori, A. Bunseki Kagaku 1938, 3 2 , 633. Horn, E. 0.; Hetllng, L. J.; Tofflemlre, T. J. Anal. N. Y. Acad. Sci. 1070, 320, 591. Safe, S.; Ztko, V. The Chemistry of PCB’s; CRC Press: Hutzinger, 0.; Boca Raton, FL, 1980. Gustafson, G. C. Envlron. Sci. Techno/. 1070, 4 , 814. Kimbrough, R. Ann. N.Y. Acad. Sci. 1979, 320, 415. f C 8 ’ s and f B B ’ s ; Monograph on the Evaluation of The Carcinogen Risk of Chemicals to Humans; Internatlonal Agency for Research on Cancer: Lyons, 1979; Vol. 18. Nlshizumi, M. a n n 1979, 7 0 , 835. Hutzinger, 0.; Sofe, S.; Zltko, V. The Chemistry of PCB’s; Chemical Rubber Co., Press: Boca Raton, FL, 1974. International Agency for Research on Cancer, Monograph 18, 1978; p
65. (35) (36) (37) (38) (39) (40) (41) (42) (43)
Jenssen, D.; Ramel, C. Mutat. Res. 1980, 7 5 , 191. Pelllzzari, E. D., private communication. Sikes, 0.; Bridges, M. Science 1952, 176. 506. McConnell, E. I n Topics In EnvironmentalHea#h;Elsevier: New York, 1980; Vol. 4, Chapter 5. Patel, C. K. N.; Burkhardt, E. G.; Lambert, C. A. Science 1974, 784, 1173. Koch, K. P.; Lahmann, W. Appi. fhys. Lett. 1978, 32, 289. Deaton, T. F.; Depatie, D. A.; Walker, T. W. Appl. fhys. Lett. 1975, 2 6 , 300. Kritchman, E.; Shtrlkman, S.; Slatkine, M. J. Opt. SOC.Am. 1978, 6 8 , 1257. West, G. A.; Barrett, J. J.; Siebert, D. R.; Reddy, K. V. Rev. Sci. Instrum. 1983, 54, 797.
J. G. Choi G. J. Diebold* Department of Chemistry Brown University Providence, Rhode Island 02912 RECEIVED for review April 21,1986. Resubmitted October 16, 1986. Accepted October 16, 1986.
Electrochromatography Using High Applied Voltage Sir: There were a few liquid chromatography experiments where an electric voltage had been applied to columns (1-5).
The term electrochromatography is defined in a book (1) as follows: “Electrochromatography should remain restricted
0003-2700/87/0359-0521$01.50/00 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
2.5KV 5 KV 11
11
t
7.4 KV
10KV
3eaker
Figure 1. Schematic diagram for electrochromatography.
to electrophoretic procedures where sorptive interaction with the support constitutes a major factor". So, in electrochromatography, sample components have been separated by both electrophoretic processes and sorptive processes with supports in modes, such as adsorption, partition, and gel permeation. Electric voltage can be applied either along or across a column. For the separation of ferritin subunits, Otsuka and Listowsky (2) applied electric voltage along a column (2 X 50 cm). By this electrochromatography, ferritin subunits were successfully separated from each other; however, the process took about 18 h. Recently, O'Farrell (3) devised a similar separation by using a gel permeation column (7 mm x 50 cm), in which BioGel A-50m (fractionation range 1OOOOO Da) and BioGel P-10 (fractionation range 1500-20000) were packed at lower and upper parts, respectively. If the solute has molecular weight, for example, around 20000, it moves a t relatively higher velocity a t the upper part compared to that at the lower part. When an electric voltage was applied appropriately (about 12 V/cm) for generating a countercurrent flow of solutes, ferritin was concentrated a t an equilibrium position adjacent to the interface of the P-10 and A-50m gel beds. Antrim et al. (5) applied voltage across the column. By use of a column packed with trimethylchlorosilane-modified carbon support material, an electric potential (from +0.8 to -0.8 V) had been applied between the support and effluent. Capacity factors of pyridine and phenol were dependent on the applied potential. The separation method, in which two functions (mobility and sorptive interaction) have been used a t the same time, might be more powerful for the separation of sample components compared to conventional liquid chromatography or column electrophoresis, if there is no difference between their mobilities or capacity factors. In this paper, we demonstrate electrochromatography by using slurry-packed microcolumns with high electric voltage along the column, up to 15 kV. In the mode of reverse-phase chromatography, a solute could be moved back and forth according to the direction of electric voltage, besides its sorptive interaction.
EXPERIMENTAL SECTION A schematic diagram is shown in Figure 1. A glass column (2 mm i.d., 10 cm length) and tetrafluoroethylenetubing (0.5 mm i.d., 7.4 cm length) packed with ODS (Deverosil,3 pm, Nomura
Kagaku, Seto, Aichi, Japan) were used as columns. Syringe injection through a septum (KF-3, Kusana Kagaku Seisakusho, Tokyo) or injection with a valve (Rheodyne7520, Cotati, CA) were used. High voltage dc power supplies (MatsusadaPrecision, Ltd., Otsu, Shiga, Japan), a UV detector (UVD-2, Shimadzu, Kyoto, Japan), and a pump (LC-4A,Shimadzu) were used. To protect the pump from damage due to high voltage, the earth lead of the dc power supply was connected to the side of the pump. As there was a high voltage gap between an outlet line after UV detector and a beaker, shown in Figure 1, the effluent was electrically sprayed into the beaker. Reagents of guaranteed grade (Wako Pure Chemical, Ltd.) were used. cis-N-Methyl-4-P-styrylpyridium iodide was a gift from K. Takagi, Nagoya University.
1~ M I N
Figure 2. Typical example of separation by applying electric voltage. Solutes were uracil and cis -N-methyl-4-P-styrylpyrMiumiodide (CSI), which were eluted in that order. For experimental conditions, see text.
RESULTS AND DISCUSSION In electrochromatography, retention time (Rt) of a solute is defined as
Rt =
L
Ru
+ u(mob)
(1)
where L, R, u , and u(mob) are column length, retardation factor (R = 1/(1+k?, k'being the capacity factor), linear flow velocity of a solute due to pressurized flow, and linear flow velocity of a solute due to the application of an electric field, respectively. The apparent linear velocity of a solute is given by Ru + u(mob). The mobility of a solute, u(mob), is given as u(mob) = u(mob)/x
(2)
where x is the applied voltage per unit length. u(mob) would be a constant value under different voltage. In the present report, the positive sign of linear flow velocity means that the flow direction is toward the column outlet, and the positive sign of applied voltage indicates that a positive voltage is applied to the side of column outlet. A typical example of separation by applying high voltage is shown in Figure 2. The applied voltages were varied from 0 to $15 kV. Electric currents were 0, 2, 4, 7, 10, and 17 MA at applied voltages of 0, +2.5, +5.0, +7.5, +lo, and +15 kV, respectively. A microcolumn (tetrafluoroethylene tubing, 0.5 mm x 7.4 cm, packed with ODS 3 gm) and an effluent mixture of methanol (88%) and 1.5 X N phosphate buffer (pH 6.7) (12%) were used. In Figure 2, we applied very high voltage to the column (up to +2 kV/cm); cis-N-methyl-4-Pstyrylpyridium iodide (CSI) was retarded significantly, although uracil was unaffected by the applied voltage due to its lack of charge. Base line separation of both solutes was obtained a t a positive applied voltage above 5 kV. Retention times vs. applied voltage is shown in Figure 3. As shown in Figure 3, the linear velocity of CSI is retarded or accelerated by applying positive or negative voltage, respectively. Ru of CSI was around 0.06 cm/s, but its dmob) was varied from +0.07 to ca. -0.07 cm/s when applied voltages were varied from -15 to +15 kV on a microcolumn (column length, 7.4 cm), respectively. In Figures 2 and 3, starting points were marked by pressing a button after every injection. An error, due to the above operation, would be included in the measurement of retention times. Therefore, for the calculation of u(mob), we used data in which k'for CSI showed larger than 1.2. The calculated value u(mob) of CSI was -2.5 x 10-~ cm2V-' s-' , and its standard deviation was 0.17 X cm2 V-' s-l. As shown in Figure 3, retention times of uracil
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 in. long, 1/4 in. diameter). Two quartz rods (11mm long, 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
