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and to simulate the conditions which might prevail in a system "with an array detector. Thermal artifacts would, of course, be smaller in the standard optical system in which the sample is illuminated with a few microwatts of narrow band light isolated by filters or monochromators.
CONCLUSION Retroreflective arrays should also be useful for compensation of refractive index gradient effects from sources other than cell heating. These include the effects of imperfect mixing of the components in gradient elution and the flow fluctuations caused by pump pulsation. The arrays should be especially useful for on-column detection and for capillary cells, since these form strong cylinder lenses that can exaggerate the effects of any refractive index gradients. The retroreflective array operates at the speed of light. The lag time is only the photon transit time from flow cell to the array and back, about a nanosecond. While we have used a 1-s time constant in our electronics, the array should operate with any achievable time constant. The performance of an absorbance detector using the phase conjugate configuration can be increased beyond its present limits. Surprisingly good results are obtained with inexpensive arrays that are not designed as high-precision optical elements. A retroreflective array can be prepared by conventional diffraction grating ruling techniques. Such a device would be more efficient than Reflexite. It should not be expensive to produce since the spacings are quite coarse and the required dimensions are small. Retroreflective arrays can certainly be used with any analytical absorption system where refractive index gradients generate problems. Obvious candidates, in addition to liquid chromatography, include flow injection analysis and atomic absorption spectrometry. Less obviously, retroreflective arrays can improve the performance of analytical spectrometric systems based on scattering or fluorescence, including optical fiber based systems. Johnson (11) has shown that inexpensive retroreflective
arrays couple light back into fibers more efficiently than plane mirrors, if the separation is greater than about 1 mm. Performance enhancements are expected in fiber systems based on absorption, fluorescence, or Raman scattering. Moreover, alretroreflective array is a simple device for refocusing source light into a fluorescence or Raman spectrometric sample cell. A retroreflective array also allows collection of light from directions away from the angle of view of the light-gathering optics. In these applications, however, high retroreflecting efficiency is more important in absorbance-based systems. Experiments designed to illustrate some of these properties are in progress.
ACKNOWLEDGMENT We thank the Reflexite Corporation for a sample Reflexite retroreflective array. Konan Peck provided useful help and suggestions. LITERATURE CITED Stewart, J. E. Appl. Opt. 1981, 2 0 , 654-659. Abbot, S. R.; Tusa, J. J. Liq. Chromatogr. 1983, 6(S-7), 77-104. Orlov, V. K.; Virnlk, Y. Z.; Vorotllln, S.P.; Gerasinov, V. B.; Kalimln, Y. A,; Asgalovlch, A. Y. Sov. J. Quantum Etectron. (Engl. Transl.) 1978, 8.
799-800.
Barrett, H. H.; Jacobs, S. F. Opt. Left. 1979, 4 , 190-192. Jacobs, S. F. Opt. Eng. 1882, 27, 281-283. Fisher, R. A., Ed. "Optical Phase Conlugation"; Academic Press: New York, 1983. Mathieu, P.; Belanger, P. A. Appl. Opt. 1980, 79, 2262-2264. Zhou, G.-S.; Casperson, L. W. Appl. Opt. 1981, 2 0 , 1621-1625. Bagdasarov, 2. E.; Virnik, Ya. Z.; Vorotllin, S. P.; Gerasimov, V. B.; Zalka, V. M.; Zakharov, M. V.; Kazanskii, V. M.;Kalinin, Yu. A.; Orlov, V. K.; Plskunov, A. K.;Sagalovich, A. Ya.; Suchkov, A. F.; Ustlnov, N. D. Sov. J. Quantum Electron. (Engl. Transl.) 1981, I 7 , 1465-1471. Gorton, E, K.; Parcell, E. W. Opt. Commun. 1983, 4 6 , 112-114. Johnson, M. Opt. Left. 1983, 8 , 593-595. Bel'dyugln, I. M. Sov. J. Quantum Electron. (Engl. Transl.) 1981, 7 7 ,
1435-1430. O'Meara, T. R. Opt. Eng. 1982, 27, 271-280.
RECEIVED for review May 20, 1985. Accepted July 19, 1985. Financial support was provided by National Science Foundation through Grant CHE-8317861.
Photothermal Refraction as a Microbore Liquid Chromatography Detector in Femtomde Amino Acid Determination Thomas G. Nolan, Brian K. Hart, and Norman J. Doviehi* Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071 Photothermal refraction, based on the crossed-beam thermal lens, is a two-laser thermoopticai technique which produces good absorbance detection limits in small probed volumes. The low detection iimlts and small probed volume of photothermal refractionsuggest Its potential for microbore liquid chromatographic detection. A simple procedure for the labeling and separation of six amino acids is employed with photothermalrefraction detectlon. Detection limits range from 5 fmoi of the DABSYL derivative of glycine to 300 fmoi of methionlne injected onto the column. Potential improvements to the technique are considered which should bring the detection limits for most amino acids to the sub-femtomoie range.
Small-bore chromatography is of value in producing low 0003-2700/85/0357-2703$01.50/0
mass detection limits (1-3). Unfortunately, a practical limitation in microcolumn liquid chromatography is the availability of suitable low-volume detectors (1-3). Although lowvolume electrochemical and fluorescence detectors have been developed ( 4 , 5 ) ,no absorbance-based detector has been developed which possesses both high sensitivity and sub-nanoliter detection volume. The combination of high sensitivity and low detection volume for conventional spectroscopic detection is limited by the path length dependence of Beer's law; an increase in sensitivity produced by an increase in path length will produce a corresponding increase in volume. Photothermal refraction, based upon the crossed-beam thermal lens, is a laser-based thermooptical detector of very small absorbance which offers a significant advantage for small volume analysis; the sensitivity of the effect is independent of path length (6-11). In this technique, a localized measurement of sample absorbance is made at the intersection 0 ID65 American Chemical Society
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region of two laser beams. Absorbance of light from a modulated pump laser beam produces a synchronous defocusing of a second, probe laser beam. Since the measurement occurs only at the intersection volume of the two beams and since the beams may be very tightly focused, very small sample volumes may be probed. Detection limits of M iron 1,lO-phenanthroline within a 10-l2-L volume have been obtained in our laboratory (11). In this paper, we demonstrate photothermal refraction for the detection of femtomole amounts of amino acids separated on a 1-mm-i.d. reversedphase HPLC column. The analysis of small amounts of amino acids is of interest in a number of biotechnologies. For example, the sequencing of rare proteins (12) or diagnosis of phenylketonuria in premature infants (13)requires the separation and detection of very small amounts of amino acids. Low picomole detection limits have been achieved by using fluorescence detection of various amino acid or protein derivatives (5,14,15). Lower detection limits would be of interest in analyzing smaller samples. Detection of amino acids using photothermal refraction is performed through precolumn formation of the DABSYL ((dimethy1amino)azobenzenesulfonyl)derivatives (16-20). These derivatives are relatively easy to form, possess a broad absorbance maximum near the 488-nm argon ion laser wavelength, and have a large molar absorptivity, over 10000 L mol-' cm-l (16). Lastly, the chromatographic separation of these amino acids has been considered in some detail (18-20).
EXPERIMENTAL SECTION Instrument. The chromatographic system is constructed from a high-pressure syringe pump (Isco Model 314), a 60-nL air-actuated injection valve (Valco Model EC14W), and a 10-cm long, 1-mm4.d. C-18 chromatography column (Isco). A mobile phase flow rate of 5.3 HL/min is typically used. The photothermal refraction system was similar to that described previously (3,with the following changes. An Ithaco Model 393 lock-in amplifier is used to demodulate the signal with a 4-s time constant. The lock-in amplifier is configured to operate in the amplitude mode; any phase fluctuations caused by temperature or flow-rate drift will not influence the measurement. A strip chart recorder is used to record the chromatograms. An argon ion pump laser is tuned to 488 nm at an output power of about 300 mW in the light-regulated mode; about 100 mW is delivered to the sample. Two microscope objectives,f = 10 and 22 mm, are used to focus the pump beam and probe beams, respectively. A mechanical chopper is used to modulate the ion laser beam at about 90 Hz. The detector cuvette consists of a 0.2-mm square-bore capillary tube (Wale Apparatus Co.), epoxied inside of a l/ls-in.-o.d. piece of stainless steel tubing. The tube is attached with a zero-volume connector to the chromatographic column. This design produces a straight flow path with minimal flow disturbance. Chemicals. DABS-CI is from Aldrich; the six amino acids are from Sigma; methanol is HPLC-grade from Alltech; and all other chemicals are reagent-grade or better. A stock pH 8.9,0.038 M buffer solution is prepared from acetic acid and sodium acetate in water. Stock amino acid solutions are prepared by dissolving a weighed amount of amino acid in the pH 8.9 buffer. A DABS-C1 stock solution is prepared by dissolving 6 mg of DABS-Cl in 10 mL of acetone. The chromatographic solvent system is 60% methanol in a pH 5, 0.05 M carbonate buffer solution. Procedure. This procedure is a combination of a reaction to prepare the labeled amino acids (20) and an extraction to separate the amino acids from unreacted DABS-Cl(21). One milliliter of the amino acid solution is placed in a test tube. The tube is placed in a beaker of hot water and the solvent is evaporated with a gentle stream of dry nitrogen gas. One milliliter of the pH 8.9 buffer and 1 mL of the DABS-Cl solution are added to the test tube. The tube is stoppered and heated to 80 "C in a hot water bath until the reaction is complete and the solution turns a light orange in color, about 5 min. A gentle stream of dry nitrogen is used
TIYE WIN)
Figure 1. HPLC chromatogram of 120 fmol of each of six amino acids. Peak 1 is glycine; peak 2 is proline; peak 3 is methionine; peak 4 is tryptophan; peak 5 is leucine; and peak 6 is phenylalanine. to evaporate the acetone from the reaction mixture. The mixture is transferred to a separatory funnel and extracted twice with ethyl ether, discarding the organic layer. The pH of the reaction mixture is adjusted near pH 3 with 0.5 M HCl. A t this pH, the reaction mixture changes color to a deep red. The DABS amino acids are extracted into ethyl ether five times. The organic layers are combined. The ether is evaporated with nitrogen. The labeled amino acids are dissolved in 1mL of the HPLC solvent mixture and filtered through a 0.22-pm filter before injection onto the chromatographic column.
RESULTS AND DISCUSSION Photothermal refraction produces a time-dependent signal whose amplitude is related to the absorbance of material located in the intersectiop region of two laser beams. Flowing samples present a particular challenge in thermooptical techniques. Flow acts to translate heat from the top to the bottom of the illuminated sample region, reducing the signal amplitude. Enhanced photothermal refraction signals may be obtained by offsetting the probe beam downstream slightly from the pump beam (9, 10). Still, it is necessary to utilize low flow rates, below 20 pL/min with a 0.2-mm square detector cell, in order to achieve a signal amplitude close to the static sample amplitude. Fortunately, these low-volume flow rates are consistent with good chromatographic separation using narrow-bore chromatographic columns. Furthermore, it is necessary to utilize a pulse-free pump since flow fluctuations produce a corresponding fluctuation in the signal amplitude. When very small amounts of amino acids are analyzed, contamination and unreacted derivatizing reagent can produce very large background peaks. In order to minimize the background contribution from the unreacted DABSYL reagent, all samples were extracted with ethyl ether. This extraction greatly reduced the background peaks at the initial portion of the chromatogram, allowing the detection of small amounts of the early eluted compounds. Figure 1presents a chromatogram of a mixture of six amino acids. The concentration of each of the acids was 2.0 X lo+ M. With an injection volume of 6.0 X lo-* L, only 120 fmol of each acid was injected onto the column. The first several peaks correspond to the blank and are presumably due to both unreacted and hydrolyzed derivatizing reagent. The instrumental response varies greatly for different amino acids, with particularly poor response for methionine, while glycine is detected with an excellent signal-to-noise ratio. The methionine response may be low for several reasons. First, it has the lowest molar absorptivity of all DABSYL amino acids studied by Lin and Chang (15),about a factor of 2 smaller
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
than glycine. Second, the extraction coefficient of methionine may be poorer than that of the other amino acids. Unfortunately, Wolski does not list the distribution coefficient for this amino acid (21). Finally, the reaction may not go to completion for all amino acids; for example, the relative responses obtained by Lammens and Verzele do not appear to be simply explainable by the variation of molar absorptivity of the different derivatized amino acids. The reaction does appear to be quantitative for glycine since a linear calibration curve was obtained a t all concentration levels investigated. A linear calibration curve, r > 0,9999, was constructed for glycine based upon peak height from the detection limit to 6X mol injected. No degradation in performance was noted at the highest concentration. The detection limit, 3a, was 5 fmol injected on-column. Of course, much less amino acid was present within the detection volume at the detection limit. Assuming a 1-pL probed volume, there were about 600 glycine molecules within the intersection region of the pump and probe laser beams. Detection limits are larger for the other amino acids: about 20 fmol for phenylalanine, leucine, and tyrosine; 10 fmol for proline; and 300 fmol for methionine. The mass detection limits are good; however, the concentration detection limit for glycine is only 8 X lo-* M. Lower concentration detection limits could be obtained by injecting larger amounts of sample onto the column. For example, a 5 - p L injection volume should produce little degradation in chromatographic performance with a 2 order of magnitude improvement in concentration detection limit. We are using a very low volume injection valve for compatibility in future work with narrower bore chromatographic columns. The sensitivity of photothermal refraction offers the interesting property of being independent of path length; strictly speaking, detection limits should be reported not in terms of absorbance but instead as the concentration-absorptivity product. For glycine, this detection limit is about 5 X lo4 cm-l. On the other hand, it is interesting to consider the absorbance across the detector cuvette at the detection limit, corresponding to an absorbance of 1 x Considering our definition of detection limit, this absorbance level is better than any other reported absorbance technique (22). Furthermore, if the path length of the measurement is defined as the pump beam spot size, 2 pm, then the absorbance detection limit is improved by 2 orders of magnitude. Although a 2-pm detector would be difficult to fabricate, we are beginning to work with a 50-pm square-bore detector. Eventually, it may be possible to employ photothermal refraction to on-column detection for very narrow capillary columns. The separation of the individual amino acids is only fair; the column produces about 1500 theoretical plates at the 5.3 pL/min flow rate. Clearly, it would be desirable to analyze more complex mixtures of amino acids at femtomole levels. A higher efficiency column will be required to resolve more complex mixtures. The chromatography of Lammens and Verzele suggests that 17 amino acids may be separated with
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a column of approximately twice the efficiency of our current column. We are currently developing longer and narrower bore columns to achieve a larger number of theoretical plates and lower mass detection limits. A factor of 3 narrower column should produce a factor of 10 improved mass detection limit. Also, additional resolution should allow separation of the early eluting amino acids from the unreacted derivatizing reagent, potentially eliminating the extraction step in our procedure. Further improvements should come from increased pump laser power. Hopefully, it will be possible to analyze most amino acids at sub-femtomole levels in a relatively simple system.
ACKNOWLEDGMENT D. Bornhop of the University of Wyoming is aLknowledged for his assistance in construction of the chromatographic system. Registry No. Glycine, 56-40-6;proline, 147-85-3;methionine, 63-68-3; tryptophan, 73-22-3; leucine, 61-90-5; phenylalanine, 63-91-2.
LITERATURE CITED Novotny, M. Anal. Chem. 1981, 53, 1294A-1308A. Scott, R. P. W. J. Chromatogr. Sci. 1980, 18,49-54. Jorgenson, J. W.; Guthrle, E. J. J . Chromatogr. 1983, 255,335-348. Slais, K.; Krejci, M. J . Chromatogr. 1982, 235,21-29. Murray, G. M.; Sepanlak, M. J. J. Liq. Chromatogr. l983, 6, 93 1-938. Dovlchl, N. J.; Nolan, T. G.; Weimer, W. A. Anal. Chem. 1984, 56, 1700-1704. Nolan, T. G.; Weimer, W. A.; Dovichi, N. J. Anal. Chem. 1984, 56, 1704-1 707. Burgl, D. S.; Nolan, T. G.; Risfelt, J. A.; Dovichi, N. J. Opt. Eng. 1984, 2 3 * 756-758. Weimer, W. A,; Dovichl, N. J. Appl. Opt., in press. Weimer, W. A.; Dovlchi, N. J. Appl. Spectrosc., in press. Nolan, T. G.; Dovichi, N. J. IEEE Circuits and Devices Magazine, in press. Deyl, Z. J. Chromatogr. 1978, 127, 91-132. Trefz, F. K.; Erlenmaier, T.; Hunnernan, D. H.;Bartholome, K.; Lutz, P. Ciin. Chim. Acta 1979, 99,211-220. Burbach, J. P. H.; Prins, A.; Lebouille, J. L. M.; Verhoef, J.; Witter, A. J. Chromatogr. 1982, 237 339-343. Lidofsky, S. D.; Imasaka, T.; Zare, R. N. Anal. Chem. 1979, 51, 1602-1604. Lin, J. K.; Chang, J. Y. Anal. Chem. 1975, 47, 1634-1638. Chang, J. Y. Anal. Blochem. 1980, 102,384-392. Chang, J. Y.; Lehmann, A.; Wittmann-Liebold, 9. Anal. Biochem. 1980, 102, 380-383. Lin, J. K.; Lai, C. C.: Anal. Chem. 1980, 52,630-635. Lammens, J.; Verzele, M. Chromatographia 1978, 11, 376-378. Wolski, T.; Go1kiewicz;W.; Bartuzi, G. Chromatographia 1984, 18, 33-36. Harris, T. D. Anal. Chem. 1982, 54,741A-745A.
RECEIVED for review May 28,1985. Accepted July 11, 1985. This work was funded by the National Science Foundation, Grant CHE-8415089, and the donors of the Petroleum Research Fund, administered by the American Chemical Society. T.G.N. gratefully acknowledges an American Chemical Society Analytical Division Fellowship, sponsored by Dow Chemical Go.
