Anal. Chem. 1986, 58,215-220
concentration of Nz(A3Z1,+) will only be beneficial if the atomizer used produces the analyte in the atomic state. In cases where the sample is largely molecular, but elemental analysis is desired, such as in chromatography detectors, it will most likely be desirable to utilize chemically reactive active nitrogen, like that produced in a low-pressure microwave discharge.
Registry No. SOz, 7446-09-5; H2S, 7783-06-4; SFs, 2551-62-4; PCl,, 7719-12-2; NaH2P04, 7558-80-7; NaZNPO4,7558-79-4; KH2P04, 7778-77-0; Na5P,010, 7758-29-4; NS, 24568-60-3; PN, 17739-47-8; Nz, 7727-37-9; N, 17778-88-0. LITERATURE CITED (1) Li, Maoliang; Fllby, R. H. Anal. Chem. 1983, 55, 2236. (2) Mlyazaki, A,; Kimura, A,; Umezakl, Y. Anal. Chim. Acta 1981, 727, 93. (3) Genna, J. L.; McAninch, W. D.; Reich, R. A. J . Chromatogr. 1982, 238, 103. (4) Na. H. C.; Niemczyk, T. M. Anal. Chem. 1982, 54, 1839. (5) Dodge, W. B., 111; Allen, R. 0. Anal. Chem. 1981, 53, 1279. (6) Capelie, G. A.; Sutton, D. 0. Appl. Phys. Left. 1977, 30, 407. (7) Rice, G. W.; Richard, J. J.; D’Silva, A. P.; Fassel, V. A. Anal. Chem. 1981, 53, 1519. (8) Sutton, D. 0.; Westberg, K. R.; Melzer, J. E. Anal. Chem. 1979, 5 7 , 1399. (9) Melzer, J. E.; Sutton, D. G. Appl. Spectrosc. 1980, 34, 434. (10) Sutton, D. G.; Melzer, J. E.; Capelle, G. A. Anal. Chem. 1978, 50, 1247. (11) D’Silva, A. P.; Rice, G. W.; Fassel, V. A. Appl. Spectrosc. 1980, 34, 578. (12) Rice, G. W.; D’Silva, A. P.; Fassel, V. A. Appl. Spectrosc. 1984, 38, 149.
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(13) Loeb, L. B. “The Kinetic Theory of Gases”; Dover Publications: New York, 1961. (14) Berkowitz, J.; Chupka, W. A.; Kistiakowski, G. B. J. Chem. Phys. 1958, 25, 457. (15) Lewis, E. P. Phys. Rev. 1904, 78, 125. (16) Hays, G. N.; Oskam, H. J. J . Chem. Phys. 1973, 59, 1507. (17) Brennen, W.; Gutowskl, R. V.; Shane, E. C. Chem. Phys. Lett. 1974, 2 7 , 138. (18) Kurzweg, U. H.; Broida, H. P. J. Mol. Spectrosc. 1959, 3 , 388. (19) Stedman, D. H.; Setser, D. W. J . Chem. Phys. 1969, 50, 2256. (20) Hays, G. N.; Tracy, C. J.; deMonchy, A. R.; Oskam, H. J . Chem. Phys . Lett. 1972, 74, 352. (21) Ung, A. Y.-M. Chem. Phys. Left. 1975, 32, 193. (22) Murai, H.; Yagi, T.; Obi, K.; Tonaka, I . Chem. Phys. Lett. 1979, 61, 513. (23) Westbury, R. A.; Winkier, C. A. Can. J . Chem. 1980, 38, 334. (24) Smith, J. J.; Jolly, W. L. Inorg. Chem. 1965, 4 , 1006. (25) Pannetier, G.; Goudmand, P.; Dessaux, 0.;Tavernier, N. J . Chim. Phys. Phys. Chlm. Biol. 1964, 67, 395. (26) Safrany, D. R. I n “Progress in Reaction Kinetics”; Jennings, K. R., Cundall, R. B., Eds.; Pergamon: New York, 1972;Voi. 6. (27) Peyron, M.; Lam Thank M. J . Chlm. Phys. Phys. Chim. Blol. 1967, 64, 129. (28) Brown, R.; Winkler, C. A. Can. J. Chem. 1960, 38, 334. (29) Jacob, A.; Westbury, R. A.; Winkier, C. A. J . Phys. Chem. 1988, 70, 4066. (30) Stevens, R. K.; Mulik, J. D.; O’Keefe, A. E.; Krost, K. J. Anal. Chem. 1971, 43, 827-831. (31) Niemczyk, T. M.; Hood, W. H.; Yang, H. C. Paper No. 1145,The Pittsburgh Conference and Exposition, New Orleans, LA, March 1985. (32) Bett, J. A.; Winkier, C. A. J. Phys. Chem. 1984, 68, 2735. (33) Herzberg, G. “Spectra of Dlatomic Molecules”; Van Nostrand Reinhold: New York. 1950;p 33. (34) Wiles, D. M.; Winkler, C. A. J. Phys. Chem. 1957, 67,902.
RECEIVED for review August 2,1985. Accepted September 10, 1985.
Pulsed Laser Thermal Lens Spectrophotometry for Flowing Liquid Detection Scott L. Nickolaisen and Stephen E. Bialkowski* Department of Chemistry and Biochemistry, UMC 03, Utah State University, Logan, Utah 84322
The pulsed laser thermal lens spectrophotometry slgnal for flowing llquld samples Is studied as a function of solution flow rate. A sllght decrease In the signal wlth lncreaslng flow rate Is attrlbuted to the competltlon between the finite rate of thermallzatlon of absorbed energy and the rate at which heat Is removed from the monltored region of the detectlon cell. The slgnal varlance Is found to be relatlvely Independent of flow rate and is llmited by source nolse and detector shot noise. The mlnlmum observable absorbance Is measured to which corresponds to 0.12 ng of aqueous 4.8 be 4.7 X pM 2-mercaptopyrldlne In the lrradlated volume. A model Is described that qualltatlvely descrlbes the flow rate dependent data In the case where lamlnar flow occurs.
The use of laser spectrophotometric techniques as detection systems for liquid chromatography (LC) and flow injection analysis (FIA) has become an area of significant research emphasis (1,2). In small-bore flow systems the small volumes, high linear flow rates, low analyte levels, and large background absorbances all represent problems in the detection and quantification of the analyte species. The special properties of laser light sources can be used to a great advantage in these
flowing systems. Two properties of laser light sources that are of importance here are spatial wave front characteristics and high spectral brightness. The wave front characteristics allow for focusing of the light beam into a diffraction-limited spot. This in turn allows delivery of the optical energy into a small volume. Nonapertured illumination of detection cells with the same transverse dimensions as the small-bore tubing can thus be accomplished. The high spectral brightness allows for maximum energy delivery at the appropriate wavelength. This property ensures maximum signals from detection schemes where a result of the analyte excitation is measured. One such detection scheme is laser excited fluorescence. The special properties of lasers have recently resulted in flowing sample limits of detection reported in numbers of molecules in a carefully designed cell (3). Detection of nonfluorescent analytes represents a particularly difficult challenge in the analysis of flowing liquid samples. Laser excitation sources have been used to an advantage here as well. Ultrasensitive laser measurements do not measure the attenuation of light as in conventional absorption spectrophotometry, but rather a secondary effect derived from the absorption of radiation is monitored. Two such techniques that have been used for the detection of flowing analytes are photoacoustic (PAS) (4,5) and thermal lens (TLS) spectro-
0003-2700/86/0358-0215$0 1.50/0 0 1985 American Chemlcal Society
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photometries (6-14). These two techniques have about the same absorbance sensitivity for solution-phase media. The high spectral brightness and spatial wave front characteristics of laser excitation sources are especially important in these techniques, since signals are related to the absorbed power per volume element. TLS (15) has been implemented by several authors for flowing sample detection (6-13). The latter studies used the chopped continuous wave (CW) laser configuration to excite the analyte. The CW configuration has been shown to exhibit a significant decrease in the TLS signal at linear flow rates greater than 3 cm/s (6). This decrease is due to the long rise time of the CW laser TLS signal relative to the time required for mass transfer and solution mixing. The flow of analyte through the cell has two effects on the formation of the thermal lens. First, laminar flow will remove heated material from the excited region of the detection cell before the TLS signal can reach its maximum value. The signal will be substantially degraded when the solution is flowing at a linear rate sufficient to sweep the heated sample out of the exciting laser beam on the time scale of the characteristic signal rise time constant. For laminar flow across the laser beam axis, this limiting flow rate may be estimated by w/t,, where w is thelaser beam radius and t, is the characteristic time constant. Thus for a beam waist radius of 0.1 mm and a t, of 20 ms, a limiting transverse flow rate of 0.5 cm/s is calculated for the CW laser TLS detector. Second, the turbulence within the cell has the effect of mixing the heated material with the surrounding material more quickly, which will also decrease the TLS signal as well as increase the signal variance because of the resulting density gradients. Pulsed laser excitation may be used to an advantage in TLS detection of flowing samples. The ratio of the theoretical enhancement factors of pulsed laser TLS to CW laser TLS is (15-17) 1.2993,
Pcwtc where E, is the energy of the pulsed laser, P C w the power of the CW laser, and t , is the time constant defined as wO2/4K, where wo is the beam waist radius a t the focus and K is the thermal diffusivity of the carrier phase. Dovichi and Harris have suggested that the effect of turbulent flow is to increase the effective thermal conductivity and subsequently decrease t , (6). Subsequently, pulsed laser excited TLS should have an advantage over that of the CW laser technique for flowing liquid analysis. Further, when using low duty cycle pulsed lasers, convective effects, which limit the sensitivity in CW laser excited TLS of static samples, will not influence the pulsed laser signals (18). This work examines the use of TLS in which a pulsed nitrogen laser is used as the excitation source, and a CW laser is used to probe the photothermal lens formed in aqueous solution. One limitation to the use of TLS is that it yields low signals when water is used as the solvent (17). The advantage to pulsed laser excitation at wavelengths around 330 nm, using available lasers, may help overcome this problem. Small pulsed nitrogen lasers (337 nm) that deliver 1-mJ pulse energies can be compared to CW HeCd lasers (325 nm) with powers typically around 5 mW. For a t, of 10 ms, a 100-fold increase in signal should be obtained with the pulsed laser for nonflowing samples. However, the main advantage to the use of pulsed laser excitation in TLS detection of flowing samples is due to the fast signal rise time, which is reflected in the inverse t, dependence of the enhancement ratio (6,7 ) . Rise and decay characteristics of pulsed laser TLS signals have been described previously (14-17,19-21). The signal rise time is limited to the slower of either the acoustic tran-
sition time, the thermalization time, or the excitation laser pulse width. In these experiments, the zero flow rate rise time is found to be due to the rate of thermalization and is approximately 10 ps. This short signal rise time should be much faster than heat transfer effects caused by thermal conduction and by turbulence or bulk flow of material through the cell. On the other hand, the decay characteristics of the pulsed laser excited TLS signal is similar t'o that for CW excitation. The pulsed laser signal decays according to (1 2t/tJ2, and the CW signal time-dependent factor is (1+ 2t/t,)-l. Although the pulsed laser TLS signal decays faster than the CW one, it is still affected by the flow of analyte (14). Subsequently, signal decay time information can still be used to determine solution flow parameters. In this study, the analytical signal is defined as the maximum value of the transient signal. This signal should be relatively independent of the carrier-phase flow rate at the moderate flow rates used for LC and FIA. Pulsed laser excited TLS should therefore be more suitable to analysis of aqueous solutions with ultraviolet excitation.
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EXPERIMENTAL SECTION The basic optical setup for two-laser TLS has been described previously by a number of authors (14-21). In this study, the pump laser was a Laser Energy, Inc., Model N2-50, coaxially excited nitrogen laser operating at a wavelength of 337.1 nm and delivering about 20 pJ of energy to the sample cell in a IO-ns pulse. Pulse repetition rates from 3 to 30 Hz were used. The l / e electric field radius of the nitrogen laser at the sample cell was measured by using a razor blade on a micrometer-driven translational stage. By use of this method a focus spot radius of 0.36 mm was measured. The probe laser was a Coherent Radiation 5-mW helium-neon (HeNe) laser. Spatial overlap of the nitrogen and HeNe laser beams was accomplished by modifying the Laser Energy, Inc., Model 337 dye laser optics so that the two lasers passed anticollinearly through the center of the flow cell. The counter-propagating 632.8-nm HeNe laser beam was separated from scattered 337.1-nm radiation of the nitrogen laser at the dye laser prism dispersion device usually used for wavelength tuning. This dispersion device consisted of a series of three fused silica prisms that resulted in a 0.174-rad exit angle difference between the HeNe beam and the scattered Nz radiation. The spatially separated beam was then directed through an OG-590 absorption filter, a 0.264 mm radius pinhole, and the detector. The HeNe laser spot at the pinhole was elliptical with dimensions of about 1 X 2 cm diameter. The signal detector used was an EG&G Model SGD-040-A photoconductive PIN photodiode. A Tektronix Model AM-502 different?al amplifier was used to amplify the signals from the photodiode. This amplifier was dc coupled and operated at maximum bandwidth. The amplified signal was recorded with a Physical Data Model 522A, 20-MH2, 8-bit transient digitizer. Multichannel averaging was used to make high S/N transients for time-resolved measurements. The nitrogen laser pulse energy was monitored by a Motorola Model MRD-500 photovoltaic PIN photodiode and amplified by an ac coupled LM741C Norton integratingamplifier. The nitrogen laser signal was digitized with an ADAC Corp. Model 1012,12-bit, programmable gain analogto-digital (A/D) converter. The transient digitizer and the A/D converter were interfaced to a DEC Model LSI 11/23 based microprocessor. Timing of the nitrogen laser pulse, transient digitizer trigger, and A/D convert command was controlled with a series of trigger delay modules. In some experiments, signal amplification was performed with an LM741C differentiating amplifier. The ac input coupled with the low gain bandwidth product of this amplifier resulted in reduced bandwidth amplification of the transient signal. As a result, noise due to acoustics and turbulance in the flowing sample was amplified with much less gain than the transient rise of the signal, and pulse-by-pulse signal measurement with an enhanced S/N could be obtained. In this detection scheme the transient was recorded and transferred to the computer where the base line and maximum values of the transient were calculated by using a pseudo-gated detection process. The analytical signal was calculated as the differencebetween the maximum value and the
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a
k
B
c
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FLOW RATE (rnL/rnin)
Flgure 1. Relative TLS signal vs. flow rate for about 0.04 mM solution of 2-mercaptopyridine in water.
base line. This signal was corrected for variations in the pulseto-pulse energy of the nitrogen laser by means of a computer program that divided the difference signal by the A/D converter signal. Because of the reduced measurement bandwidth, this detector amplifier could not be used when examining the temporal signal behavior. The flow cell was constructed from stainless steel and was fitted with fused silica windows spaced 4 mm apart. This cell had an effective volume of about 0.3 cm3. The detection cell window normal was placed at a 55O angle to the direction of laser propagation to minimize reflective losses. This resulted in an optical path length of 0.7 cm through the solution phase. Focusing of the N2laser into the center of the cell was accomplished with an f/3 concave mirror, whereas the HeNe laser was focused in front of the cell center with an f/3 lens. Solution flow was perpendicular to the laser beam propagation direction. Flow of the aqueous solutions through the cell was accomplished by maintaining a positive pressure of nitrogen gas over the solution reservoir. The flow rate was controlled by adjusting the gas pressure above the reservoir and was measured as a volume flow rate using a graduated cylinder. Filtration of the flowing solution with Whatman GF/A glass filters was performed in-line prior to the detection cell. Tubing made of Teflon (1.5 mm i.d.) was used throughout the system. The analyte used for the flow study was 2-mercaptopyridine. This compound was chosen because it has a low fluorescence quantum yield when excited at the wavelength of the nitrogen laser, using water as the solvent. Molar absorbances of highconcentration stock solutions were obtained with a Varian series 634 UV-vis spectrophotometer.
RESULTS AND DISCUSSION The relative nitrogen laser pulse energy corrected TLS signal is plotted in Figure 1. The flow rate of the analyte solution was varied from 0 to 11mL/min. These volume flow rates are higher than are typical for LC or FIA (1,2,6, 7), but because of the large cross section area of the cell used in these experiments, the linear flow rates are on the order of those that would be found in microcells commonly used in LC detectors. The line drawn through the experimental data is the regression line obtained without the zero flow rate points. These data are a collection of data sets taken over a several-week period with each set obtained by using a slightly different apparatus configuration. The different apparatus configurations were a result of “fine tuning” of the optics. Each point in this figure represents an average of 500 data points, and each set of 500 laser pulse averaged data is scaled relative to the zero flow datum. In contrast to the flow rate dependent decrease in the signal of the single CW laser configuration (61, the signal of the pulsed laser configuration shows no significant decrease as the flow rate increases. This is in agreement with the fast rise time of the thermal lens relative to heat and mass transfer effects and is also observed
TIME (MSEC) Figure 2. Tlme-resolved TLS signal for about 0.04 mM solution of 2-mercaptopyridine at flow rates of (A) 0.0 mL/min, (B) 2.3 mL/min, (C) 4.7 mL/min, and (D) 9.0 mL/min. These data were obtained by averaging 500 single pulse transients and correcting for the background signal.
for the case of flowing gas analysis (14). The decrease of the zero flow signal over that a t finite flow may be due to the effects of static temperature build up within the cell (14,15) or photolysis of the analyte. A static temperature increase results in changes in the overall thermal conductivity, density, refractive index, etc., of the solution, as well as a static thermal lens and an analyte density gradient due to the solution density gradient. The analyte concentration can also vary along the temperature gradient, as in the Soret effect (22). All of these factors will influence the signal magnitude in the absence of solution mixing. The formation of the static temperature increase distribution is highly dependent on the repetition rate of the pulsed excitation laser and the particular design of the cell. If a cell were designed to incorporate a very small sample volume and a large thermal conductivity, these static thermal effects would be minimized. However, if photolysis is taking place, only flowing the analyte can remedy the signal decrease. Time-resolved signals taken at several different flow rates of aqueous solutions are shown in Figure 2. These data were obtained at a pulse repetition rate of 28 Hz and with a dilute sample. Multichannel averaging was used. Each transient illustrated is an average of 500 pulses of the background, obtained with the N2laser beam blocked, subtracted from that from 500 pulses of the signal. The reproducible background interference was primarily due to the radio frequency noise generated by the N2 laser discharge and resulted in a slight “ringing” artifact in the uncorrected signal transients. The corrected data are scaled such that the maximum signal is 1. This scaling allows for direct comparison of the signal rise and decay characteristics. The signal rise and decay times are dependent on flow rate. The greater the flow rate, the faster are the rise and fall times. The zero flow rate data are linear when plotted in such a fashion as to reflect the characteristic time constant. If the signal rise time is neglected, the signal is proportional to the inverse focal length of the thermal lens, and the focal length in turn is proportional to (1 2t/tC)2. A characteristic time constant of t , = 16.5 ms is found. By use of the thermal diffusivity of water, K = 0.001 456 cm2/s, and the time constant, an effective focus spot size of the N2 laser of 0.1 mm is calculated. With the effective beam waist calculated above, the acoustic transition time limit is 67 ns. However, the zero flow data exhibit a rise time of about 10 hs. Subsequently, the signal rise time is probably limited by the time required for thermalization of the absorbed radiation. Weimer and Dovichi have recently shown that with kinetic modeling of the flow rate dependent CW laser excited TLS
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signals, the precision of the measurement could be increased and the characteristic constants could be obtained directly from the modeled transients (23). Flow rate and absorbance were both determined in their model regression analysis. Subsequently, modeling of the data was used to compensate for the diminished signal due to analyte flow. However, the precision of this derived value would still decrease with increasing flow rate. In the present experiments, there is no large change in the signal magnitude or variance with increasing analyte flow. However, the effective decay constant changes significantly. Kinetic modeling could be used as a sensitive method for determining the flow rate, and this value could in turn be used to compensate for any change in the signal magnitude as a function of analyte flow rate. There are two ways by which the effective decay characteristics of the pulsed laser TLS signal can change with analyte flow (6). The first is by turbulent mixing in the illuminated region of the sample. This mixing will result in an effective decrease in t,, which is proportional to the flow rate. The second is due to laminar flow. Laminar flow across the illumination axis will result in a translation of the thermal lens across this axis and a subsequent decrease in the decay time. The pulsed laser TLS signal can be expressed in terms of the focal length of the thermal lens (15,17,20,21,24). This focal length is in turn calculated from the temperature distribution
”( $) *6T(r, t )
l/f = no
P
dr2
where 1 is the length of the monitored region assumed to be less than the Rayleigh range of the focused excitation beam, no is the refractive index of the solution, and (dn/dT), is the change in refractive index with temperature at constant pressure, which is primarily due to the change in density accompanying the temperature change. With the use of (dn/dT), in eq 2, it is implicitly assumed that the acoustic transition time is much less than the time required for any of the important kinetic processes, e.g., thermal diffusion. 6T is the temperature change associated with the excitation and subsequent thermal relaxation of the sample. The spatial and temporal temperature change is calculated by using the Greens function integral for thermal diffusion. For a temperature change that is Gaussian in profile and is formed by a firstorder kinetic process after the relatively short-pulsed excitation, the temperature change can be expressed as 6T(r, t ) = exp[-(t - t’)/t,] (1 2t‘/tc) exp[-2r2/w2(1 2 t ’ / t c ) ] dt’
?A
+
+
where t, is the time constant for energy thermalization, LY is the exponential absorption coefficient, p is the density, and C, is the heat capacity. Substitution of eq 3 into 2 yields exp[-(t - t’)/t,]
4r2
w 2 ( 1 + 2t’/t,)
X
-
J
- 1 exp[-2r2/uO2(1+ 2t’/t,)]dt’ (4)
In static experiments all terms involving the off-axis radius, r, are neglected since the resulting thermal lens remains on axis with the collinear laser beams. But for a flowing sample with laminar flow transverse to the optical axis, the entire thermal lens will move with some linear velocity, u,away from
2.5-
0
2
TIME
I
I
I
6
E
10
(rnsec)
Figure 3. Plot of eq 4 with t , = 16.5 ms and wo = 0.01 cm. Flow rates are as follows: (A) 0.0 cm/s, (B) 1.0 cmls, (C) 2.0 cm/s, (D) 5.0 Cm/s, (E) 10.0 cm/s, and (F) 20.0 cm/s.
the beam axis. The linear flow rate is actually a function of position in the cell (23). Points closer to the cell walls and windows will have a lower flow rate than those in the cell center. However, because of the complex design of the cell used in these experiments, calculation of the position-dependent flow rates is not possible. For purposes of a qualitative description we may assume that the radius used to evaluate the inverse focal length is only a function of time and flow rate, r = ut. Substitution of this into eq 4 gives a description of the pulsed laser TLS signal for flowing analyte samples. Figure 3 shows the results of numerical integration of eq 4 evaluated for the t,, w,,, and t, determined from the zero flow rate data illustrated in Figure 2. Equation 4 was evaluated at a number of linear flow rates. Only the time-dependent inverse focal length was determined. There will also be a contribution to the signal that is due to photothermal deflection (24). However, since the pinhole size was much less than the beam waist at the detector and since the gradient of a Gaussian beam is zero at the beam center, contribution to the signal as a result of beam deflection is negligible with the small thermal gradients generated in these experiments. In the calculations, the increase in the rate of signal decay at higher flow rates is not due to a change in the characteristic time constant at increased flow. Rather it is due to the fact that the off-axis radius, r, in eq 4 changes with time due to the translation of the thermal lens in laminar flow. The slight decrease in the TLS signal seen at higher flow rates in Figure 1 can also be explained by examining eq 4. As the signal decay rate increases due to increased linear flow, competition between signal rise and decay occurs. A t higher flow rates the signal will begin to decay before its maximum can be reached, resulting in decreased signal strength. This can be seen in Figure 3 where the focal length maximum decreases as the linear flow increases. But for substantial decrease to occur would require very high linear flow rates, which are unlikely for LC or FIA analyses. The calculated time-dependent signals appear very much like the experimental data in Figure 2. The data illustrated in Figure 4 can be used to compare the model to the experimental data. In this figure, the effective t , are plotted as a function of linear flow rate for both the experimental and calculated transient signals. For the experimental data, t, was determined from the slope and intercepts of the inverse square root signal as a function of time, as described above for the zero flow rate data. However, the inverse square root signals for the faster flow rates, above 4 cm3/min, were not linear over the entire 10-ms range, and the t , were determined from the first 5 ms of the transient signals for these data. The source
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+ o +
c+ f
-2
I
0
I
2
I
4
I
6
I
e
I
10
12
LINEAR FLOW RATE (CM/SEC) Figure 4. Comparison of the experimental t , (+) to that of the calculated (0) based on the laminar flow model. The experimental flow rates are scaled by a factor of 66.7 om-*.
of this nonlinearity may be seen by comparison of the calculated transient signals to those obtained experimentally. The calculated transients decay to a zero signal level, whereas the experimental data obtained at the faster flow rates decay to a fiiite signal level, which exhibits a slower decay time. This latter effect may be due to the cell location dependent flow rate. In this case, the slower decay would be due to that part of the solution close to the cell windows, and thus it does not move as fast as the bulk of solution. Scaling of the flow rate axis in Figure 4 was performed by visual inspection of the experimental t,. The scaling factor used was 66.7 cm-2, which is on the order of the scaling fador for the tubing used in these experiments. This scaling factor corresponds to a tubing inner diameter of 1.4 mm. The experimental t, were also examined to see if the rate of decay was linearly dependent on flow rate as would be the case if turbulent mixing were to cause an effective increase in the rate of thermal conduction. To test this, a plot of the inverse t, vs. volume flow rate was examined for linearity. It was found that the data did not fit this model since the resulting plot exhibited a large curvature. Finally, we have examined the flow pattern in the cell by observing the path followed by small air bubbles that occur in solution when the flow is initially started. The flow appears to be confined to the middle of the cell with a minor amount of circular flow in the two “D”-shaped cavities on either side of the line between the inlet and the outlet ports. This, along with the evidence above, indicates that the laminar flow model is at least qualitatively correct. The relative standard deviations of the data illustrated in Figure 1were up to 11% . This high variance occurs even with compensation for the nitrogen laser pulse energy. Since the signal variance is relatively constant over the flow rate range studied, this variance is not due to flow rate dependent turbulence (6),but rather it is due to source noises inherent in the apparatus. Source noise variance is due to short-term power variations and pointing noise of the HeNe probe laser, as well as mode variation and pointing noise in the nitrogen laser. However, measurements made of the noise characteristics of the HeNe laser indicate that it is only a minor source of signal variance. The main source of this noise may be mode instability and pointing noise of the nitrogen laser. The signal variance, and thus the measurement precision, could be improved by spatial filtering of the excitation beam. Another source of variance is detector shot noise. This noise results in a single pulse signal-to-noise ratio of 2 at an absorbance of 3.9 X Measurement errors due to shot noise were further aggravated by the gated sampling procedure. The relative shot noise is dependent on the particular detector and pinhole size used. But for all practical purposes, it is a con-
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stant. Improvements must come by enhancement of the signal. There are two realistic means by which the signalto-noise ratio can be improved first is the use of smaller f / # optics for focusing of the excitation laser. In pulsed laser TLS, the enhancement factor is proportional to w0-4 (17). Thus a decrease in the f / #will result in both an increase in the signal and a decrease in the excited volume. Both of these enhancements are useful in LC and FIA analysis. The second means by which the signal level can be increased is to increase the excitation energy. In the present experiments, only 10% of the laser energy was delivered to the cell. Using fewer optical elements and using coated optics would result in nearly a 10-fold increase in signal. Clearly, this and perhaps other experimental optimizations must be performed before this method is to be useful as a LC detector. One of the objectives in LC detector design is to have a low variance so that a small amount of analyte can be observed in the presence of a large background signal. This objective is not met with the current design. The sensitivity of the TLS apparatus is ultimately limited by the minimum absorption of the analyte that will produce an observable signal. The TLS signal was measured at concentrations ranging from 1.0 to 0.020 mM and was found to be linear with respect to the analyte concentration. The calculated minimum detectable signal for a signal-to-noise ratio of 4 after 500 averages corresponded to a concentration of 4.8 FM 2-mercaptopyridine. This in turn corresponds to a minimum observable absorbance of 4.7 X and a minimum mass limit of 0.12 ng of the analyte in the irradiated volume for 337.1-nm irradiation. The minimum detectable absorbance found in this work is not very close to the figure of estimated from flowing gas-phase signals and enhancement factor ratios for water and argon gas (14). In these studies the excitation source was not at the maximum in the absorption profile of the analyte. Smaller detection limits niay be observed by utilizing tunable laser excitation sources. Analyte-specific detection may also be accomplished by varying the wavelength of the excitation laser. By this means, species with an absorption peak at the specific excitation wavelength would be detected while those with no significant absorption would pass by undetected.
CONCLUSION The technique of pulsed laser TLS as a flowing solution phase detector is potentially very useful in trace analysis in flowing samples and as a LC detector. The applicability is nearly universal by the use of tunable dye lasers tuned to the optimum excitation wavelength of the species to be analyzed. Pulsed laser excited TLS appears to be superior to the CW technique for flowing sample analysis. The pulsed laser apparatus has the advantages of a signal magnitude that is relatively insensitive to flow rate, a rapid rate of data aquisition, relative immunity to thermal convection effects due to a low duty cycle and a low average power, relative flow rate independent data precision, and an enhancement factor that is greater than that for the CW technique. However, because of the poor precision due to pulsed laser pointing noise and mode variations, much more work must be performed before this detection method will become practical. We are currently working on methods to minimize these source noise limitations. LITERATURE CITED (1) Yeung, E. S. Adv. Chromafogr. ( N . Y . ) 1984, 23, 1-63. (2) Green, R. Anal. Chem. 1983, 55, 20A-32A. (3) Dovlchi, N. J.; Martin, J. C.; Jett, J. H.; Trkula, M.; Keller, R. A. Anal. Chem. 1984, 56, 348-354. (4) Tam, A. C. In “Ultrasensitive Laser Spectroscopy”; Kliger, D. S., Ed.; Academic Press: New York, 1983; pp 2-98. (5) Sawada, T.; Oda, S. Anal. Chem. 1981, 53, 539-540. (6) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1981, 53, 689-692. (7) Leach, R. A.; Harris, J. M. J . Chromafogr. 1981, 278, 15-19.
Anal. Chem. 1986, 58,220-222
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(8) Sepaniak, M. J.; Vargo, J. D.; Kettler, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 54, 1252-1257. (9) Buffett, C. E.; Morris, M. D. Anal. Chem. 1982, 54, 1824-1825. (IO) Buffett, C. E.; Morris, M. D. Anal. Chem. 1983, 55, 376-378. (11) Morris, M. D. R o c . S f I E I n t . SOC. Opt. Eng. 1983, 426, 116-120. (12) Leach, R. A.; Harris, J. M. Anal. Chem. 1984, 56, 2801-2805. (13) Pang, T. J.; Morris, M. D. Appl. Spectrosc. 1985, 39, 90-93. (14) Nickoiaisen, S. L.; Blalkowski, S. E. Anal. Chem. 1985, 57,758-762. (15) Fang, H. L.; Swofford, R. L. I n “Ultrasensitive Laser Spectroscopy”; Kliger, D. S., Ed.; Academic Press: New York, 1983; pp 178-233. (16) Long, G. R.; Bialkowskl, S. E. Anal. Chem. 1984, 56, 2806-2811. (17) Mori, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1982, 54, 2034-2038. (18) Buffett, C. E.; Morris, M. D. Appl. Spectrosc. 1983, 37, 455-458. (19) Siebert, D. R.; Grabiner, F. R.; flynn, G. W. J . Chem. Phys. 1974, 60, 1564-1574.
(20) (21) (22) (23) (24)
Barker, J. R.; Rothem, T. Chem. Phys. 1982, 68, 331-339. Twarowskl, A. J.; Kliger. D. S. Chem. Phys. 1977, 20,253-258. Gigiio, M.; Vendramlni, A. Appl. fhys. Lett. 1974, 25,555-557. Welmer, W. A.; Dovlchi, N. J. Anal. Chem. 1985, 57, 2436-2441. Jackson, W. B.; Amer, N. M.; Boccara, A. C.; Fournier, D. Appl. Opt. 1981, 20, 1333-1343.
RECEIVED for review April 22, 1985. Resubmitted August 5, 1985. Accepted September 30, 1985. This research was supported in part by BRSG SO7 RR07093-17 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.
Optical Sensor for Oxygen Based on Immobilized Hemoglobin Zhang Zhujun’ and W. Rudolf Seitz* Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
The oxygen sensor consists of a 0.5 mm thick layer of deoxyhemoglobin immobilized on catlon exchange resin and positioned on the common end of a bifurcated fiber optic bundle. An 0,-permeable TFE Teflon membrane separates the knmobiilzed reagent phase from the sample. The sensor Is based on the shlft in the Soret absorption band of hemoglobin upon association with 0,. The specific parameter measured Is the ratio of reflected intensities at 435 and 405 nm. This parameter may be used to measure 0, partlal pressure from 20 to 100 torr H a suitable cailbratlon curve is established. Reflected intensitles and Intensity ratlos decrease with the amount of Immobilized hemoglobin. For a reagent layer 0.5 mm thick, the optimum hemoglobin loading is 0.03 g/g catlon exchange resin. I t takes approximately 3 min to reach steady state when an 0, sample is introduced. The sensor must be stored in a reducing medium to prevent oxidation of hemoglobinto methemoglobin. I t Is stable for 2 days when stored at room temperature and for a week when stored at 4 OC.
We have prepared an optical sensor for oxygen based on immobilized hemoglobin. The sensor exploits the shift in the Soret absorption band when deoxyhemoglobin combines with oxygen to form oxyhemoglobin. The ratio of the hemoglobin reflectances a t 405 and 435 nm serves as a measure of 0 2 partial pressure. One attractive feature of this approach is that it involves a true equilibrium. In contrast, the oxygen electrode requires steady-state mass transfer to 0, to the electrode surface and thus is subject to error if factors which affect 0, mass transfer are not properly controlled (1). The same is true for an 0 2 sensor based on chemiluminescence (2). Optical 0, sensors based on fluorescence quenching avoid this problem but are not true equilibrium sensors because response depends on the relative rates of fluorescence and nonradiative return to the ground state via interaction with 0, (3-6). Thus this type of sensor is sensitive to slight changes in medium, which affect fluorescence, and to the presence of other quenchers. Present address: Department of Chemistry, Shaansi Normal University, Sian, Shaansi, People’s Republic of China.
A more fundamental advantage of the approach described here is that it is based on a spectral shift. Because the oxygenated and deoxygenated forms each have distinct spectra, there is inherently more information available than there is from a fluorescence quenching based sensor where the measured parameter is a decrease in intensity. The most serious limitation of the O2sensor based on immobilized hemoglobin is that its useful lifetime is short because hemoglobin degrades. A second problem is that the measured response function is nonlinear with O2 partial pressure and is not readily described mathematically.
EXPERIMENTAL SECTION Apparatus. The apparatus for the O2sensor is similar t o
apparatus used earlier in our laboratory for pH and pC0, sensors (7,8). The output of a 250-W tungsten halogen lamp (Edmund Scientific) is passed through one of two dielectric interference filters with peak transmittances at 435 nm and 405 nm and bandwidths of 6.8 nm and 9.2 nm, respectively, at half-maximum transmittance. The filtered beam from the lamp is passed through one arm of a bifurcated fiber optic bundle (common end diameter 4.5 mm) to a thin layer of immobilized hemoglobin. The reflected intensity is monitored through the other branch of the bifurcated fiber optic bundle by an RCA 1P21 photomultiplier tube. The photocurrent is displayed on a Spex digital photometer (Model DPC 2) and recorded on a Heath SR-255B strip chart recorder. Figure 1shows a detailed view of the reagent phase and sample cell. A piece of Tygon tubing holds a 25 pm thick TFE Teflon membrane (American Durafilm Co., Inc.) on the end of a 7 mm i.d., 50 mm long piece of glass tubing. The membrane serves to separate the sample from the reagent phase, which consists of an immobilized suspension of deoxyhemoglobin in phosphate buffer. Samples are introduced or withdrawn by syringe. 0thenvise the cell is sealed to prevent ambient O2from entering. The sample cell is covered with A1 foil on the outside to enhance the reflected signal (a tactic that will only work if the sample is transparent at 405 and 435 nm). In operation, the entire sample cell assembly is covered with black cloth to exclude ambient light. Absorption spectra were obtained with a Shimadzu Model UV-200s double-beam spectrophotometer. A Matheson Model 7401T gas proportioner connected to nitrogen and oxygen cylinders was used to prepare standard oxygen solutions. The pH was measured with an Orion digital Ionanalyzer/SOl equipped with an Orion combination glass pH electrode. Reagents. Hemoglobin (substrate powder type 11), sodium dithionite and CM-Sephadex-C-50-120 were purchased from Sigma Chemical Co. Oxygen (99.6%) and nitrogen (99.7%) were
0003-2700/86/0358-0220$01.50/0 0 1985 American Chemical Society
