504
Anal. Chem. 1986, 58,504-505
of As(II1) and As(V). Both manifolds produced the same signal for a given As(II1) concentration. The selectivity increases expected, as described above. for the addition of KI in the second dual phase manifold were observed. Further increases in selectivity through the use of KI/NH20H remain to be investigated.
(9) Welz, B.;Melcher, M. Analyst (London) 1984, 109, 577. (10) Narasaki, H.; Ikeda, M. Anal. Chem. 1984, 56, 2059. (11) Yamamoto, M.; Yasuda, M.; Yamamoto, Y. Anal. Chem. 1985, 5 7 , IRR9
ikeda,
(12) M. Anal. Chim. Acta 1985, 167,289. (13) Astrom, 0. Anal. Chem. 1082, 5 4 , 190.
G. E. Pacey* M. R. Straka J. R. Gord
LITERATURE CITED Straka, M. R.; Pacey, G. E.; Gordon, G. Anal. Chem. 1984, 56, 1973. Straka, M. R.; Gordon, G.; Pacey, G. E. Anal. Chem. 1985, 57, 1799. Hollowell, D. A,; Pacey, G. E.; Gordon, G. Anal. Chem. 1985, 57, 285 1. Van der Linden, W. E. Anal. Chlm. Acta 1983, 151,359. Attlyat, A. S.; Chrlstlan, G. D. Talanta 1985, 3 1 , 463. Dedina, J. Anal. Chem. 1982, 54,2097. Welz, B.;Melcher. M. Ana/yst (London) 1984, 109, 569. Welz, B.; Melcher, M. Analyst (London) 1984, 109, 575.
Department of Chemistry Miami University Oxford, Ohio 45056
RECEIVED for review September 19,1985. Accepted November 21, 1985.
AIDS FOR ANALYTICAL CHEMISTS Slmple Nanoliter Refractive Index Detector Darryl J. Bornhop and Norman J. Dovichi* Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071 Lasers are convenient light sources for optical methods of analysis. I s particular, the spatial coherence of the laser is useful when probing small volume samples. For example, probe volumes in the nanoliter range have been reported for fluorescence (1-41, absorbance (5-9), and light scatter ( 1 0 , l l ) measurements. The push for small-volume detectors comes from the development of narrow-bore liquid chromatography (12-16), where submicroliter samples must be detected to achieve the full separation power of liquid chromatography. Further interest in small-volume optical techniques arises from biological analysis of small amounts of rare substances (17). The refractive index detector is one of the few universal optical detectors available for liquid samples. Unfortunately, the sensitivity of refractive index measurements is inherently low; the refractive indexes of most solutes differ only in the second or third decimal place. It is not suprising that state of the art refractive index detectors tend to be rather complex. For example, detection limits of An = 1.5 X have been reported using an interferometric measurement of optical path length, proportional to the refractive index of the solute (18). Unfortunately, the high-quality Fabry-Perot interferometer used in this refractive index detector is quite expensive. Furthermore, the interferometric refractive index design produces a signal that is proportional to path length. To achieve the excellent detection limits, a 10-cm path length and 2OO-pL probed volume was required. Scaling to submicroliter volume would require a significant decrease in path length, producing a proportional decrease in sensitivity. In this paper, we wish to describe a simple, inexpensive, and low-volume refractive index detector. This detector is based on the propagation of a beam of light through a liquid-filled tube as shown in Figure 1. The fluid-filled tube may be considered by analogy to a lens; an off-axis ray passing through a lens is both defocused and deflected by the lens. The deflection and defocusing is quantitated as a change in the laser beam intensity measured by a small-area photodiode located near the inflection point of the beam profile. These measurements are taken relatively far from the cuvette. Of course, the small diameter of the cuvette suggests that a simple model based upon a lenslike behavior is a gross 0003-2700/86/0358-0504$01.50/0
simplification. Diffraction and reflection from the edges of the tube and aberrations induced by the tube will act to distort the laser beam profile. Several models exist for the propagation of a light beam through a pair of concentric tubes with differing refractive index. These models arise in the determination of the refractive index profile of a clad optical fiber or single mode preform (1S22). We note that Jorgenson has proposed an on-column detector for capillary liquid chromatography that is based upon the scattering of light by the fluid-filled capillary (14).
EXPERIMENTAL SECTION Optical System. A schematic of the optical system is shown in Figure 2. The system is constructure on an optical table, NRC Model KST-48. A 5-mW helium neon laser, Melles Griot Model 05LHP151, provides a linearly polarized beam at 632.8 nm. A microscope slide is used to split a small portion of the beam to a reference detector, described below. The main beam is focused by a 16-mm focal-length microscope objective, Melles Griot, into the round Pyrex capillary sample cell, 0.5 mm i.d., 0.7 mm o.d., Wale Apparatus. The tube is mounted on two stacked translation stages that provided sample positioning both along and perpendicular to the laser beam. Retroreflections from the tube can produce unwanted feedback into the laser resonator. We tilt the tube by about 20° to eliminate these retroreflections. This laser produces very low drift in intensity. However, detecting very small reflective index changes requires very stable intensity measurements. To reduce long-term, low-frequencydrift, we utilize a double beam in space optical design (23). The reference and signal detectors are identical 1-mm2silicon photodiodes, Silicon Detector Model SD 041-11-11-011. The output of the photodiodes is conditioned with matched current-to-voltage converters, consisting of a JFET operational amplifier, Linear Devices LF 351, wired with a 1-Ma feedback resistor in parallel with a 47-pF capacitor. The signal and the reference photodiode outputs are subtracted with an instrumentation amplifier, Analog Devices Model AD 524. This circuit allowed for the removal of common-mode intensity fluctuation in the beam, primarily line frequency noise, in addition to providing 10 times signal gain. A piece of frosted glass is located before the reference detector. Translation of the frosted glass is a convenient way to adjust the intensity of light reaching the reference detector, which facilitates nulling of the dc signal component. The signal is next sent to 0 1986 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
Figure 1. Deflection of a light ray by a fluid-filled tube. The hatched cylinder represents the glass tube that holds the fluid.
Figure 2. Experimental dlagram. The laser is a polarized hei1um:neon laser: B, Is a microscope slide beam splitter; the lens is a 16-mm focal-length microscope obJect1ve:the cuvette is a 0.5 mm i.d., 0.7 mm 0.d. tube; D1 and 0 2 are matched 1-mm2 photodiodes with matched current-to-voltage converters; and I A Is an Instrument amplifier used to subtract common-mode laser noise from the deflection signal. A 5 '/,digit voltmeter is used to record the signal.
an active low pass filter with a cutoff frequency of 50 Hz. Finally the resulting voltage is displayed on a Keithley Model 195/1950 digital multimeter. One hundred readings are recorded automatically at 1-s intervals by the multimeter. The mean and standard deviation are computed for data gathered at the 1-s intervals. Alignment. The following alignment produces a large deflection signal. Small adjustments about these positions should optimize the signal. The cuvette is located about 22 mm past the lens and 0.4 mm off the laser beam axis. The photodiode is located about 10 mm past the cuvette. The profile of the laser beam at the detector face is elliptical and non-Gaussian. A relatively sharp dividing line exists between the intense beam center and an adjacent dark fringe. The dividing l i e moves along the major axis as the refractive index of the material within the tube is changed. The small area photodiode is located at this dividing region between the light and dark beam areas. The optimum detector position is found by measuring the voltage difference obtained between water and the methanol solution ab a function of the detector position along the laser beam major axis. The beam intensity changes by about a factor of 70 between water and a 1:l water/methanol solution for a well-aligned system. It should be noted that many weak light and dark fringes are observed far from the beam center along the major axis of the laser beam. These fringes undergo a very large change in position as the sample refractive index is varied. A detector could be located at theae fringes to increase the sensitivity of measurement. Currently, our measurement appears to be limited by temperature drift of the system, which produces a concomitant refractive index change of the analyte. Reagents. All chemicals are reagent grade or better. Alignment is performed by using deionized water and a 50% water/ methanol solutiofi. For calibration, a 10% glycerol/water (w/v) stock solution is prepared. From this stock solution, 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, OB%, LO%, and 1.2% solutions are used to construct a calibration curve. R E S U L T S A N D DISCUSSION
A calibration curve was constructed from aqueous glycerol solutions. The calibration curve is linear, r > 0.9997, from the detection limit, 2a, of 6 x lo-' to at least 1.4 X IOw3in units of ARI. Higher concentration samples appear to produce a smaller than expected signal. The detection limit is determined for a 1-s sampling rate and is dominated by low frequehcy drift in the system, probably caused by temperature fluctuation in the room; the refractive index of most liquids
505
is a function of temperature. The change in refractive index with temperature for water, -8 X ' V I , is smaller than for most liquids (24). The detection limit corresponds to a "C/s. The cuvette temperature fluctuation of about 7.5 x is attached to a relatively massive aluminum block, which helps attenuate temperature drift. Further sources of noise arise from air flow past the laser beam. The air flow acts both to change the temperature of the sample and also to perturb the propagation properties of the laser beam. We enclosed the entire system inside a large box to eliminate air flow. Another noise source is due to vibration of the optical components. The entire system is secured with massive fixtures to the well-damped optical table. The detection volume of the cuvette is quite small. The intersection volume of the 10 pM radius laser beam with the 250 pM radius sample tube is about 200 pL. A more practical detection volume is given by the area of the tube times its radius, 60 nL. About 400 pg of glycerol is present at the detection limit. Several improvements in the system heed to be investigated. First, the intensity-sensitive detector could be replaced with a position-sensitive detector. These devices, based upon several designs, produce a signal directly proportional to the deflection of the laser beam. Since they are immune to intensity fluctuations, they could produce improved detection limits. The use of a position-sensitive detector would eliminate the need for the differential optical design. Second, the temperature of the system needs to be stabilized. We have briefly investigated the performance of the system for flowing samples. Slightly improved detection limits are produced, apparently due to the large thermal mass of the high-pressure syring pump utilized in the experiment. Third, smaller diameter cuvettes need to be investigated. The simple model based upon the lenslike nature of the fluid-filled tube suggests that smaller diameter cuvettes will produce higher sensitivity signals. These cuvettes would prove useful in modeling oncolumn capillary liquid chromatography detection. Registry No. Glycerol, 56-81-5. LITERATURE C I T E D (1) Diebold, G. J.; Zare, R. N. Science 1977, 796, 1439-1441. (2) Hirschfeld, T. Appl. Opt. 1978, 15, 2965-2966. (3) Folestad, S.;Johnson, L.; Josefsson, B.; Gaile, B. Anal. Chem. 1982, 5 4 , 925-929. (4) Dovichi, N. J.; Martin, J. C.; Jett. J. H.; Keiler, R. A. Science 1983, 219, 845-847. ( 5 ) Pelietier, M. J.; Thorsheim, H. R.; Harrls, J. M. Anal. Chem. 1982, 5 4 , 239-242 -.. - -
(6) Nolan, T. G.; Weimer, W. A.; Dovichi, N. J. Anal. Chem. 1984, 5 6 .
1704-1707. (7) Burgi, D. S.;Nolan, T. G.; Risfelt, J. A.; Dovichi, N. J. Opt. Eng. 1984, 2 3 , 756-758. (8) Weimer, W. A.; Dovlchi, N. J. Appl. Opt., in press. (9) Nolan, T. G.; Dovichi, N. J. Anal. Chem., in press. (10) Hercher, M.; Hueller, W.; Shapiro, H. M. J . Histochem. Cytochem. 1979, 2 7 , 350-352. (11) Zarrin, F.; Dovichi, N. J. Anal. Chem. 1985, 5 7 , 1826-1829. (12) St. Claire, R. L.; Jorgenson, J. W. J . Chromatogr. Scl. 1985, 2 3 , 186-191. (13) Rokushika, S.;Yln Qiu, 2.; Hatano, H. J . Chromfitogr. 1985, 320, 335-342. (14) Jorgenson, J. W.; Guthrie, E. J. J . Chromatrogr. 1983, 255, 335-348. (15) Tsuda, T.; Tanaka, I.; Nakagawa, G. J . Chromatogr. 1982, 239, 507-513. (16) Novotny, M. Anal. Chem. 1981, 5 3 , 1294A-1308A. (17) Beersma, D. G. M.; Hoenders, B. J.; Huiser, A. M. J.; van Toorn, P. J . Opt. SOC. Am. 1982, 7 2 , 583-588. (18) Woodruff, S. D.; Yeung, E. S. Anal. Chem. 1982, 5 4 , 1174-1178. (19) Presby, H. M.; Marcuse, D.; French, W. G. Appl. O p t . 1979. 78, 4006-40 11. (20) KokLlbun, Y.; Iga, K. Appl. Opt. 1980, 19, 846-851. (21) Cooper, P. R. Appl. Opt. 1983, 2 2 , 3070-3072. (22) Ball, S. Opt. Eng. 1985, 2 4 , 518-521. (23) Weimer, W. A.; Dovichi, N. J. Anal. Chem., in press. (24) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1979, 5 1 , 728-731.
RECEIVED for review July 25, 1985. Accepted September 6, 1985. This work was supported, in part, by a University of Wyoming, College of Arts and Science, Basic Research Grant. r
