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Anal. Chem. 1985, 57,2700-2703
Liquid Chromatography Absorbance Detector with Retroreflective Array for Aberration Compensation and Double Pass Operation Teng-Ke Joseph Pang and Michael D. Morris* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
A retroreflecting array Is used In a llquld chromatography detector for dynamlc compensation of aberratlons caused by thermally induced refractlve Index gradients and for performIng a second pass through the cell. Use of the array In the phase conjugate conflguratlon decreases the base line noise In the system to the level of source intensity fluctuations. The double pass geometry Increases the absorbance by a factor of 2. Overall, slgnal/nolse lntensltles are Increased by a factor of 4-6. Prospects for further Improvement are dlscussed.
The requirements for optical design of a cell for a low-noise absorbance detector for liquid chromatography are well understood (1, 2). A long light path is needed to maximize absorbance and sufficient light throughput is necessary to make shot noise low. These requirements must be satisfied in a design in which the cell is not overfilled. Light striking the structure of the cell heats it. The heat leads to slowly fluctuating refractive index gradients. The gradients function as lenses which cause the amount of light passing through limiting apertures beyond the cell to vary. That effect produces a wavy base line. It becomes increasingly difficult to satisfy cell design requirements as the cell volume becomes much less than 1pL. Cells of 0.05-2.4 p L are often constructed as cylinders of about 0.25-1 mm diameter and 1-3 mm length. Other designs use a capillary tube, usually 0.25-1 mm i.d., illuminated perpendicular to the length of the capillary. The same geometry is encountered in on-column detectors, which have been proposed for use with capillary columns. All of these cells are easily optically overfilled, and all have shorter path lengths than is desirable. In the present communication we describe an optical configuration which simultaneously doubles the optical path length and compensates for the noise generated by overfilling the cell and heating the cell structure. For this purpose we use a retroreflective array functioning as an approximatephase conjugator (3-5). Phase conjugation has been widely investigated for compeneation of aberrations in coherent optical systems (6). A generalized optical system for phase conjugation is shown as the upper diagram of Figure 1. In the phase conjugate configuration the light source, which need not be a point source, is at 0 and an observer or detector views the corrected pseudo-image of the source at D. In this system the light wave, U, is transformed by additional phase changes to U’ as it propagates through a perturbing medium. The perturber can be any optical element or combination of elements, such as lenses. The perturber may also include less controlled elements such as refractive index gradients caused by heating the perturber or keeping it in motion. The perturbed wave interacts with the phase conjugator to generate a new wave, U’*. This phase conjugate wave
propagates in exactly the opposite direction to Uc. The wave U’* can be described mathematicallyas the complex conjugate of U’. This property is the origin of the name of the phenomenon. As the phase conjugate wave traverses the path of U’ back toward the source of U, the original perturbations to the wave are exactly undone. After passage through the perturbing medium, the complex conjugate of the original wave, U*, is obtained. U* can be separated from U with a beam splitter and observed at point D. Commonly the system is arranged so that the distance 0-P is equal to the distance D-P. In this case the phase conjugate image of an extended original object will look exactly like the original. Because the phase conjugate image is not generated from the light source directly, it is often called a pseudo-image. A variety of nonlinear optical effects can be used to generate phase conjugate waves. However, these all require that the light source be a laser, and many require that the laser have high instantaneous power as well. Further, some have slow response times, on the’order of seconds. Such phase conjugators would appear to have limited utility for aberration compensation in liquid chromatographic systems. Orlov and co-workers (3) and, independently, Barrett and Jacobs (4)demonstrated that an array of corner cube reflectors functioned as an approximate phase conjugator. The Orlov configuration used a specially constructed optical element (3). Barrett and Jacobs (4) used inexpensive replica arrays, designed for use in self-illuminated highway signs. They pointed out that these devices did not require coherent illumination and could even function with white light. Barrett and Jacobs coined the term “pseudo-conjugator” to describe the device. Several groups have employed retroreflective arrays as distortion compensating mirrors in laser resonators (7-10). Johnson (11) has investigated retroreflective arrays as reflectors on the moving objects in fiber optic interferometric Doppler velocimeters. O’Meara (13) has reviewed the properties and potential applications of several classes of approximate phase conjugators in aberration compensation and has described several possible array configurations based on reflecting or refracting elements. The theory of retroreflective arrays has been outlined (4, 12,13). The governing equation is formally identical with the diffraction grating equation. The retroreflected light appears in multiple orders (4). For a sufficiently large number of elements, the array directs most of its light directly back to the source. The lowest order contains most of the reflected intensity. The phase conjugate configuration automatically provides two passes through the perturbing medium. With a retroreflective array, both passes are made by the same light wave. Viewed as a spectrophotometer this configuration provides automatic double passing through a sample. It follows that the absorbance measured in the retroreflected beam should be twice the absorbance observed in a single pass through the same sample.
0003-2700/85/0357-2700$01.50/00 1985 American Chemical Society
4NALYTICAL CHEMISTRY, VOL. 57, NO. 13. NOVEMBER 1985 * 2701
I
D u*T
AVPLIFIER
0 B E N ? SPLITTER
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"
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Flgure 2. Conventional absorbance detector: N.D., neutral density fiiter; other components as in Figure 1.
of inexpensive retroreflective arrays will be inferior to devices constructed for spectroscopic purposes.
R R A
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SAMPLE
CfLL
BEAM SPLITTER
FWre 1. Upper diagam, th5 phase mjqate configurafion. The li@t sowce generates wave U from point 0. phase perturbations transfcm this to U'. m e phase conjugate wave, U". is generated by the phase conjqator at point P and is transformed to U' by passage through the perturber. The phase conjugate wave, U', is observed at point D.
Lower diagram, absorbance detector employing retroreflective array as approximate phase conjugator: R.R.A., retroreflective array: B.S., 8020 beam splitter: P.M.T., photomultiplier tube: L, 50 rnrn focal length lens: R. load resistor; radiation source: Ar+ laser or xenon arc lamp. The phase conjugate configuration requires that the output light he transmitted to the detector or viewer through a beam splitter. If the beam splitter transmits fraction f of the incident light, then the detector sees fraction 1- f. I t follows that the light incident on the detector is reduced to the fraction f(l- fl of the intensity transmitted in a straightthrough configuration. A 5050 beam splitter maximizes the detected intensity which can be obtained from a system including a retroreflective array. This value is 25% of the intensity of the light which would be transmitted through a straight-through system of equivalent path length. The further loss of detected intensity is less than a factor of 2 for beam splitters in the range 1 5 8 5 t o 8515. It is understood that a retroreflective arrav does not function as a perfect phase conjugator (4,5,12,13). However, for many kinds of aberrations, among them refractive index gradients formed by local heating or turbulence, compensation is quite good. Compensation is best if the gradient extends over a distance which is large compared to the element-toelement separation in the array. Commercially available retroreflective arrays have some disadvantages as elements in spectrophotometric optical systems. They are sold primarily as materials for constructing self-illuminated highway signs or warning markers to he affixed to clothing or vehicles. For these applications, fairly coarse arrays are satisfactory, and the quality of the optical surfaces need not be high. Consequently, the performance
EXPERIMENTAL SECTION The experimental apparatus for conventional absorbance measurements is shown as Figure 2. The lower diagram of Figure 1 shows the modifications for use in the phase conjugate configuration. A 0.2-m monochromator (Instruments SA, Model H-20) with a 1200 groove/mm grating and 1-mm slit was used to achieve a 4-nm band-pass. A 1P28 photomultiplier was used as a detector. The light source was modulated by a mechanical chopper (Laser Precision CTX-534) a t 12.7 Hz and demodulated with a lock-in amplifier (PARC 186) operated with a 1-s time constant. Modulation served as a convenient means to identify the desired signal in the presence of significant light intensity reflected from various components in the optical system. Data were recorded on a small computer equipped with a 12-bit analog-digital converter. In the conventional absorbance configuration, the light was attenuated hy neutral density filters before introduction into the monochromator. An 8020 glass beam splitter was used in the phase conjugate configurationto deliver 20% of the retroreflected beam to the monochromator. The retroreflective array was a small sample (approximately 2-cm square) of Reflexite (ReflexiteCorp) fastened to a sheet of aluminum. The Reflexite was positioned approximately 20-25 cm from the exit window of the absorbance cell. The Reflexite was mounted so that its surface was perpendicular to the light path in the system. Two light sources were used an argon ion laser (Lexel85-1), operated at 458 nm with output power attenuated to 2-5 mW with neutral density filters, and a 100 W xenon arc lamp (Ealing Corp.), mounted in a housing fitted with a condensing lens. The entire available output from the lamp was focused on the absorbance cell. The absorbance at 462 nm was measured. The absorbance cell used was a 0.5-pL volume, 1 mm path length flow cell (Kratos SFA-234),connected to the outlet of a liquid chromatographic column. The column was 4.6 X 250 mm packed with 10-pm Lichrosorh RP-18. The pump was a dual piston type (LDC Mini-pump) fitted with a pulse dampener and operated at a flow rate of 1.4 mL/min. A 1-pL sampling loop was used to inject the sample onto the system. The solvent was 80% methanol (Burdick & Jackson) and 20% deionized water. The test samples were o-nitroaniline (Aldrich), prepared as 22 g/L solutions. Data were taken at 1-s h t e ~ a l s .The photoeurrent values were converted to absorbance, using the average photocurrent during the solvent-onlyportions of the chromatograms to define 100% transmittance. Noise was calculated as the root mean square value of the absorbance fluctuation measured over a 30-9period. RESULTS AND DISCUSSION Chromatograms taken with the optical system in the conventional and phase conjugate configurations are shown as
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985 No pseudo-phase
conjupotion
No p8eudo-phose
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0.120 0.1 10 0.100 p)
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e
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c8 2
0.050 0.040 0.030 0.020 0.010 0.000 -0.01 0
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0.280 0.260 0.240 0.220 0.200 0.180 0.160 0.140 0.120 0.100 0.080 0.080 0.040 0.020 -
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Figure 4. Chromatograms of 0-nitroanliine with arc lamp source: upper curve, conventional absorbance detector; lower curve, detector with retroreflectivearray.
Figure 3 and 4. Figure 3 shows chromatograms taken with laser illumination and Figure 4 with arc lamp (incoherent) illumination. The optical elements were deliberately positioned so that the cell would be visibly overfilled. Inspection of the figures shows the performance improvements obtained with the phase conjugate configuration. With both coherent and incoherent illuminationthe double pass behavior of the phase conjugate configuration was confirmed. The absorbance in the phase conjugate configuration was always twice the absorbance in the single pass configuration, within 5% or less. This error is quite close to the overall experimental error in our rather crude optical system. The absorbanceratio A,,/A, is 0.78, in good agreement with the value (0.77) measured independently on a commercial spectrophotometer. With laser illuminationthe absorbance is doubled while the base line noise is reduced from about 0.0032 absorbance unit root mean square to about 0.0011, The overall S I N is enhanced by a factor of 6.1. With the incoherent source the signal is also doubled while the noise is reduced from 0.0033 to 0.0014 absorbance unit. The overall SIN improvement is a factor of 4.1. It is difficult to assess the exact improvement in signalto-noise ratio brought about by the retroreflective array. With both coherent and incoherent illumination, the noise was reduced to the noise in the source intensity itself. The lower performance with the incoherent source appears to be due largely to the crude output regulation of the available xenon lamp system. The lamp was subject to random variations in output, causing base line drift and jumps in the chromatograms. However, it is clear that the signal-to-noise ratio
improvement in the phase conjugate configuration is significant and dose not depend on the use of coherent light. Better performance could be obtained with a system employing a carefully regulated light source. We have measured the reflectance of Reflexite at several wavelengths in the 450-650-nm region. At the distances used in these experiments the reflectance is 20-21 % . The reflectance depends somewhat on distance because the number of elements illuminated by an uncollimated beam is distancedependent. We observed significant light scattering from the Reflexite under all conditions. The scattering is largely due to the poor optical quality of the surfaces of the array. Because the reflectance of Reflexite is only about 20%, the reflected light from the surfaces of the sample cell is a significant fraction of the total intensity seen by the detector. Modulating the retroreflected beam with a mechanical chopper adequately distinguishes it from cell surface reflections. Alternatively, a flow cell with angled or curved windows could be used so that the surface reflections are directed away from the detector. We have attempted to make measurements in the ultraviolet region. However, the low efficiency of the available monochromator in this region and our reliance on a glms beam splitter and lenses frustrated these attempts. Theory (4,12, 13) predicts that the performance of array should be only weakly wavelength-dependent. A system constructed with quartz optics and a monochromator with a grating blazed for UV operation should perform satisfactorily. For these experiments, we have used the reverse optics configuration (broad band source, monochromator after the flow cell) both to exaggerate the thermal artifacts in the cell
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Anal. Chem. 1905, 57,2703-2705
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 electrochemicaland 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
