Development of a Conductivity-Based Photothermal Absorbance

Jun 27, 2006 - ... University of North Carolina at Chapel Hill, Venable Hall, CB#3290, .... current-to-voltage converter, rectifier, and low-pass filt...
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Anal. Chem. 2006, 78, 5309-5315

Development of a Conductivity-Based Photothermal Absorbance Detector for Capillary Separations Stephen E. Johnston,† Keith E. Fadgen,‡ and James W. Jorgenson*

Department of Chemistry, University of North Carolina at Chapel Hill, Venable Hall, CB#3290, Chapel Hill, North Carolina 27599-3290

A contactless conductivity-based absorbance detector has been developed for use with capillary separations. Detection is based on a photothermal process. As analytes pass through the detector they absorb light, producing a thermal perturbation. This thermal event results in a change in the solution conductivity. The measured change in conductivity is directly related to the absorption of light. The major advantage to this type of detector is that the measured absorbance is, to a first approximation, independent of optical path length, allowing small-diameter capillaries to be used. This approach combines the optical simplicity of traditional transmission-based instruments with the path length independence of similar refractionbased photothermal detectors. In addition to the initial development and characterization of the photothermal absorbance detector, multiphysical modeling of the heat transfer within the conductivity cell was performed. Traditional UV absorbance detection techniques, both direct and indirect, are the most common detection techniques utilized in capillary electrophoresis. These techniques, however, are limited by the path length sensitivities inherent in absorbance detection. From Beer’s law, the magnitude of absorbance observed is directly related to the path length of the probed area. As the path length decreases, as in small-diameter capillaries and microfluidic chips, the effectiveness of traditional absorbance techniques diminishes. The detection methods available for microcolumn separations are also limited due to the need to preserve the separation efficiency of the chromatography or electrophoresis. There have been several attempts to increase the path length at the point of detection. These methods have resulted in an increase in the detection capabilities, however, usually at the expense of the separation efficiency. These methods have included the development of the Z-Cell1,2 and the bubble cell.3 Both methods employed changes in the geometry of the capillary at the point of detection and led to reported increases in S/N, but the improvement is limited by the band broadening effects caused by the change in geometry. * Corresponding author. E-mail: [email protected]. Fax: (919)962-2388. † Current address: Schering-Plough Corp., 1011 Morris Ave., Union, NJ 07083. ‡ Current address: Waters Corp., 34 Maple St., Mail Stop CT, Milford, MA 01757. (1) Dasgupta, P. K.; Bellamy, H. S.; Liu, H. Talanta 1993, 40, 341-345. (2) Kahle, V. Biomed. Chromatogr. 1999, 13, 93-94. (3) Xue, Y.; Yeung, E. S. Anal. Chem. 1994, 6, 3575-3580. 10.1021/ac052223j CCC: $33.50 Published on Web 06/27/2006

© 2006 American Chemical Society

Alternative methods of absorbance detection, not requiring changes in capillary geometry, have been developed based on a thermooptical effect. Traditional absorbance systems operate by detecting changes in transmitted light and relating this back to an absorbance value. The light that is absorbed, however, results in physical changes in the solution. Within the illuminated region, an increase in temperature proportional to the sample absorbance is observed due to the nonradiant relaxation of the analyte.4 To a first approximation, thermooptical-based techniques are path length independent, allowing for their favorable use with microcolumns. One of the more researched thermooptical effects is the change in refractive index due to the rise in temperature. In 1984, the initial theory5 and experimental results6 were published by Dovichi et al. for a laser-induced photothermal refraction system. Pump and probe lasers were focused perpendicularly into a bulk sample. Light absorbed from the pump laser resulted in a change in refractive index, causing the angle at which the probe beam exited the sample to change slightly. The pump beam was modulated, allowing for the use of a lock-in amplifier to isolate the change in refractive index due to the absorbance of the light. This technique was soon adapted for use with capillary separations by the Dovichi group in the detection of amino acids with detection limits of ∼1 µM (0.5 fmol).7-12 The refractive index absorbance detector is not optically simple, though. Two lasers must be used and must be focused to the same spot within the capillary. Another thermooptical effect that has been explored is a change in viscosity due to the change in temperature. The temperature dependence of viscosity in aqueous solvents is an ∼2% relative change per degree temperature change.13 This is a 2 orders of magnitude larger relative change than that of refractive index (0.009%). Unfortunately, thermal changes in viscosity are not easily measurable within microcolumns. However, viscosity (4) Saz, J. M.; Diez-Masa, J. C. J. Liq. Chromatogr. 1994, 17, 499-520. (5) Dovichi, N. J.; Nolan, T. G.; Weimer, W. A. Anal. Chem. 1984, 56, 17001704. (6) Nolan, T. G.; Weimer, W. A.; Dovichi, N. J. Anal. Chem. 1984, 56, 17041707. (7) Nolan, T. G.; Hart, B. K.; Dovichi, N. J. Anal. Chem. 1985, 57, 2703-2705. (8) Nolan, T. G.; Dovichi, N. J. Anal. Chem. 1987, 59, 2803-2805. (9) Yu, M.; Dovichi, N. J. Anal. Chem. 1989, 61, 37-40. (10) Yu, M.; Dovichi, N. J. Appl. Spectrosc. 1989, 43, 196-201. (11) Bruno, A. E.; Paulus, A.; Bornhop, D. J. Appl. Spectrosc. 1991, 45, 462467. (12) Waldron, K. C.; Dovichi, N. J. Anal. Chem. 1992, 64, 1396-1399. (13) CRC Handbook of Chemistry and Physics, 51st ed.; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1970; sections D and F.

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is related to several other physical properties of the solution, including electrical conductivity. An experimental design similar to the refractive index design was used by McLaren and Dovichi to detect the change in conductivity.14 A modulated helium-neon laser was focused into a Pasture pipet into which electrodes had been inserted. The current output of the detection electrode was sent through a current-to-voltage converter, rectifier, and low-pass filter before being detected by a lock-in amplifier whose reference was set as the laser modulation frequency. A sample of methylene blue in a sodium chloride buffer was detected with a detection limit of 0.2 µM. The theoretical considerations on current were later investigated taking into account, among other things, the time constant of the thermal relaxation and the spatial profile of the laser beam.15 Presented herein is the development of a conductivity-based absorbance detector (hereafter referred to as a photothermal absorbance detector) for use with capillary separations. Initial experimental characterization of the detector will be shown along with theoretical modeling of the heat transfer within the system. EXPERIMENTAL SECTION Materials and Buffers. Sodium tetraborate decahydrate, glucosamine, and 4-dimethylaminoazobenzne-4’-sulfonyl chloride (dabsyl chloride) were obtained from Sigma (St. Louis, MO). Ammonium carbonate and ammonium hydroxide were obtained from Fisher Scientific (Fair Lawn, NJ). All compounds were used as received. Deionized water was obtained from a Barnstead Nanopure System (Boston, MA). The CE run buffer used was 20 mM borate, pH 9.3, prepared in deionized water. The buffer used in the labeling reaction was 50 mM ammonium carbonate, adjusted to pH 9.0 using ammonium hydroxide. Buffer solutions were filtered using a 0.2-µm nylon membrane filter from Alltech Associates (Deerfield, IL) and then vacuum degassed. Preparation of Dabsyl-Labeled Glucosamine. The procedure used to label glucosamine with dabsyl chloride has been described in the literature.16 Briefly, the procedure was as follows: glucosamine was dissolved in 50 mM ammonium carbonate at a concentration of 1 mM. Dabsyl chloride was dissolved in acetone at a concentration of 6 mM. Twenty milliliters of each solution was combined with the resulting mixture heated for 12 min in a 75 °C hot water bath. The solution was allowed to cool for 10 min before being evaporated to dryness under a nitrogen stream. The dried material was then reconstituted in 10 mL of deionized water. In an attempt to remove the remaining ammonium carbonate buffer, the sample was lyophilized three times, giving a final volume of 3 mL. The final sample was prepared by diluting 0.100 mL of the tagged sample to a final volume of 5.00 mL in the 20 mM borate buffer. Assuming the reaction went to completion with no sample loss, the dabsyl-glucosamine (FW 461.43) concentration in the sample would be ∼48 µM. The sample was characterized by visible absorbance in a 1.00cm cuvette on a Lambda 35 UV/Vis spectrometer (PerkinElmer, Wellesley, MA). The absorbance was 1.672 and 1.539 at 442 and 488 nm, corresponding to the output lines of a He:Cd and argon ion laser, respectively. (14) McLaren, R.; Dovichi, N. J. Anal. Chem. 1988, 60, 730-733. (15) McLaren, R.; Dovichi, N. J. Can. J. Chem. 1989, 67, 1178-1186. (16) Lin, J.-K.; Chang, J.-Y. Anal. Chem. 1975, 47, 1634-1638.

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Figure 1. Detection circuitry used for photothermal absorbance detector. The signal out was then sent to dual lock-in amplifier setup.

Capillary Electrophoresis (CE) System. CE was performed in an untreated fused-silica capillary (Polymicro Technologies, Phoenix, AZ) with an inner diameter of 50 µm, an outer diameter of 360 µm, and a length of 68 cm. Sample was injected electrokinetically at 2 kV for 4.5 s. A 30-kV dc, reversible polarity power supply (Spellman High Voltage Electronics Corp., Plainview, NY) was used in the positive mode. The electrophoresis current was monitored by measuring the voltage drop across a 220 kΩ resistor connecting the outlet buffer vial to ground. UV-visible absorbance measurements were made with a Linear UVis 200 detector (Thermo Capillary Electrophoresis, Fremont, CA) fitted with an on-capillary detection cell. Photothermal Absorbance Detector. The basis of the photothermal absorbance detector is a contactless conductivity detector described previously by our group.17 The cylinders used for electrodes were 23-gauge, 304 stainless steel hypo tubing (Small Parts, Miami Lakes, FL) drilled out to an inner diameter of 390 µm and cut to lengths of 5 (two electrodes) and 2 mm (one electrode). A dual conductivity cell was formed when the three electrodes were soldered to a printed circuit board (ExpressPCB, Redwood City, CA) with a 1.1-mm air gap between each electrode (5 mm-2 mm-5 mm pattern). The cell was broken into a reference gap and a detection gap. A 100-kHz ac voltage was applied to the middle (2 mm) electrode with the resulting current then measured at each of the outer electrodes. Similar to the contactless conductivity detector, the first circuit component connected to each electrode was a currentto-voltage converter. This was followed by a phase and gain correction circuit (Figure 1). The phase and gain of one of the measured signals was fixed, while the other was adjusted using a pair of linear potentiometers. The balanced signals were then sent to a comparator circuit with the output sent to a lock-in amplifier. (17) Johnston, S. E.; Fadgen, K. E.; Tolley, L. T.; Jorgenson, J. W. J. Chromatogr., A 2005, 1094, 148-157.

Figure 2. Experimental setup for photothermal absorbance detector.

The op-amp used in the current-to-voltage converter, and in all phase and gain corrections, was an OPA602A (Texas Instruments, Dallas, TX), which was chosen for its low current noise characteristics (0.6 fA/xHz). All resistors involved were thin-film resistors chosen for their precision and temperature stability. The area of the PCB directly behind the detection gap was removed by mill. The output of a 10-mW, 442-nm HeCd laser (model 4210B, Liconix, Sunnyvale, CA) or an adjustable power 488-nm argon ion laser (model Innova 70A, Coherent, Santa Clara, CA) was directed with a mirror toward the detection gap. The beam was sent through an SR540 optical chopper (Stanford Research Systems, Sunnyvale, CA) before being focused through a 6.3× microscope objective (Melles Griot, Irvine, CA). The focused beam was aligned on the capillary. When a line focus was desired, the chopped beam was focused through a cylindrical lens (43940, Oriel, Stratford, CT) in place of the microscope objective. The cylindrical lens was placed within an optical rotator (13011, Oriel) to allow for alignment of the focused line with the capillary axis. Signal Processing. An SR810 digital signal processor (DSP) lock-in amplifier (Stanford Research Systems) was used for signal isolation and amplification. The external reference signal was provided to the lock-in amplifier by a DS335 function generator (Stanford Research Systems). The function generator was set to output 20 Vp-p at 100 kHz and was used both as the reference for the lock-in amplifier and as the excitation source for the detector. The output of this lock-in amplifier was sent to a second SR810 DSP lock-in amplifier. The frequency of the optical chopper was set as the reference to the second lock-in. The bandwidths on each lock-in were set to allow for the frequencies of interest to pass through. The time constant on the first lock-in was set at 1 ms with a 24 dB/octave slope, resulting in a bandwidth of 159 Hz. This was a wide enough bandwidth to let the chopping frequency through. The time constant of the second lock-in was set at 100 ms, with a 24 dB/octave slope, resulting in a bandwidth of 1.59 Hz.

The use of a reference gap/detection gap setup allowed for nullification of the background signal. Nullification was necessary to prevent overloading of the second lock-in amplifier. For the 20 mM borate buffer, using a 20-Vp-p, 100-kHz input, a background of ∼490 mV was seen. After nullification, a typical background level for this setup was ∼5 mV with an rms noise of 0.5 µV. The output of the second lock-in amplifier was digitized by a PCI-MIO-16XE-50 data acquisition card (National Instruments, Austin, TX) in a personal computer. Data points were collected and analyzed on the computer by custom software written in LabVIEW (National Instruments) and Igor Pro (WaveMetrics, Lake Oswego, OR). The results from each CE run were median filtered, baseline subtracted18 prior to any calculations being performed. A schematic of the entire experimental setup is shown in Figure 2. The use of a dual lock-in amplifier system allowed for the sensitive detection of only those changes in conductivity resulting from the absorbance of light. A sample of detection using the 10mW HeCd laser is shown in Figure 3. As conductive species pass through the detector, a response similar to that of a first derivative is observed at the output of the first lock-in amplifier. For the same detection, a single analyte peak is observed at the output of the second lock-in. Although there were several conductive species traveling through the detector, only one analyte had a change in conductivity that was modulated at the same frequency as the modulated laser light. Thus, only the single analyte was identified by the dual lock-in amplifier system. RESULTS AND DISCUSSION Response to Laser Power. Experiments were originally performed using a 10-mW HeCd laser and an adjustable power argon ion laser. An improvement in S/N with increasing laser power was quickly observed, and all further experiments and data collection were performed using the argon ion laser. A sample of (18) Moore, A. W.; Jorgenson, J. W. Anal. Chem. 1993, 65, 188-191.

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Table 1. Summary of Results for the Effect of Laser Power on the Response of the Photothermal Absorbance Detector for a Separation of Dabsyl-Labeled Glucosaminea

Figure 3. Comparison of output of (A) first lock-in amplifier and (B) second lock-in amplifier for the detection of a 48 µM dabsyl-labeled glucosamine sample in 20 mM borate buffer.

power (mW)

peak height (µV)

peak area (µV‚s)

baseline noise (µV)

S/N

9.58 51.6 101 146 188 251 299

7.64 36.4 72.7 109 128 170 195

0.146 0.703 1.44 2.31 2.74 3.66 4.23

0.38 0.49 0.49 0.53 0.54 0.48 0.52

20 75 150 210 240 350 370

a Modulation frequency was set at 50 Hz. No standard deviations are given due to differences in the exact laser power used during each detection.

Table 2. Summary of Results for Effect of Modulation Frequency on Photothermal Absorbance Detection of a Separation of Dabsyl-Labeled Glucosaminea modulation peak height peak area baseline noise frequency (Hz) (µV) ((σ, %) (nV‚s) ((σ, %) (µV) ((σ, %) 50 75 100 150 175 200 225 250

Figure 4. Effect of laser power on S/N for a separation of 48 µM dabsyl-labeled glucosamine in 20 mM borate buffer. Modulation frequency was set at 50 Hz.

48 µM dabsyl-labeled glucosamine in 20 mM borate buffer was detected at laser powers from 10 to 300 mW at a constant modulation frequency of 50 Hz. Linear regression analyses were performed on the peak area and peak height data. For the peak area, a slope and intercept of 1.41((0.02) × 10-8 V‚s/mW and 5((3) × 10-8 V‚s were obtained, respectively, with a correlation coefficient (R2) of 0.997. For the peak height, a slope and intercept of 6.42((0.09) × 10-8 V/mW and 6((2) × 10-8 V were obtained, respectively, with a correlation coefficient of 0.996. Baseline noise remains fairly constant as a function of laser power for all except the lowest laser powers. Because of this, the S/N increases linearly (R2 of 0.989) with respect to laser power (Figure 4). Peak height, peak area, baseline noise, and S/N values with respect to laser power are compiled in Table 1. Although a maximum power of 700 mW was available from the argon ion laser, an experimental limit of 300 mW was observed due to scattered light hitting and burning the printed circuit board. This problem was lessened by removing a larger area of circuit board from behind the gap. For comparison, an average S/N of 181 was observed for the 48 µM dabsyl-labeled glucosamine using a commercial UV-visible detector set at 442 nm with 0.010 AUFS and a rise time of 0.1 s. The injection and capillary dimensions were identical to those used with the photothermal detection. Absorbance by the dabsyl-labeled glucosamine is slightly greater at 442 nm than at 488 nm, giving a slight edge to the UV-visible detection in that respect. Signal filtering was the same for each detection method as the detection 5312 Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

163 ((1.2) 113 ((4.3) 69.5 ((2.0) 32.0 ((0.4) 23.5 ((0.7) 15.3 ((1.1) 10.6 ((1.6) 7.38 ((1.1)

3490 ((1.4) 2500 ((2.5) 1560 ((1.3) 706 ((1.0) 533 ((1.6) 348 ((3.7) 242 ((3.3) 170 ((2.4)

0.45 ((3.7) 0.37 ((4.6) 0.27 ((7.8) 0.13 ((3.6) 0.093 ((8.0) 0.081 ((12) 0.057 ((6.6) 0.041 ((5.8)

S/N ((σ, %) 370 ((4.8) 310 ((4.8) 250 ((6.1) 260 ((3.9) 250 ((7.3) 190 ((12) 190 ((6.1) 180 ((6.8)

a Peak area and peak height values are uncompensated for the bandwidth effects of the dual lock-in amplifier system.

bandwidths were each 1.59 Hz. At laser powers greater than 150 mW, a more sensitive detection was achieved with the photothermal detector. Response to Modulation Frequency. The detector response was measured as a function of modulation frequency. The modulation frequency was varied between 50 and 250 Hz with the laser power held constant at 200 mW. Peak height, peak area, baseline noise, and S/N data are summarized for each frequency in Table 2. It was expected that an increase in modulation frequency would cause an inverse decrease in signal. However, the nature of the decrease was not entirely due to changes in conductivity. Using a 1-ms time constant with a 24 dB/decade roll-off, a 159-Hz bandwidth was in effect on the first lock-in amplifier. The use of 60- and 120-Hz line filters, however, also added to the bandwidth of the lock-in. To measure the effects of the lock-in bandwidth on the detected signal, an AD532 multiplier chip (Analog Devices, Norwood, MA) was used to determine the gain of the two lock-in amplifier system as a function of frequency. A 100-kHz, 2-Vp-p sine wave was multiplied with a low frequency (20-500 Hz), 1-Vp-p square wave using the AD532. The output of the multiplication contained a dc offset that was removed using a capacitor. The signal was then input into the first lock-in of the dual lock-in amplifier system. The first lock-in was referenced to the 100-kHz sine wave, while the second lock-in was referenced to the square wave. By varying the frequency of the square wave between 20 and 500 Hz, the gain response as a function of frequency of the dual lock-in system was determined.

Figure 5. Effect of modulation frequency on the output of the photothermal absorbance detector. (A) The compensated (O) and uncompensated (+) peak area for the detection of a 48 µM dabsyllabeled glucosamine sample in 20 mM borate buffer. (B) The uncompensated output of the first lock-in amplifier for the same detection.

Using this gain response, the peak height and area data were corrected. A comparison of the measured and corrected peak area data is shown in Figure 5A. A shallower decrease in signal was observed in the corrected data, as it no longer included the effect of the bandwidth of the detection electronics. To investigate the response at the lowest possible modulation frequencies, the frequency was varied between 4 and 140 Hz with the laser power held constant at 250 mW. Figure 5B depicts the uncorrected decline in the output of the first lock-in amplifier as a function of increasing modulation frequency. As the frequency decreased, the depth of modulation increased, and a large rise in signal was observed. Examining the conductivity change of a single modulation cycle, a sawtooth pattern was seen with two distinct rises and decays (Figure 6A). The first rise in signal was steep and short, ∼10 ms in length. The second rise was shallower and lasted the remainder of the upward rising half-cycle. However, the second rise was cut off as faster modulation frequencies were used. It is believed that the two rises and decays correspond to two periods of heating: the first the immediate heating and cooling of the solution, the second the secondary heating and cooling of the fused-silica capillary. For a single long-duration application of light, these two distinct rises were still present (Figure 6B). Fitting

Figure 6. Presence of different time constants in the heating (and cooling) of a dabsyl-labeled glucosamine solution for (A) modulated detection of (a) 4, (b) 35, and (c) 105 Hz and (B) single manual application of the laser.

exponentials to the rises and decays, time constants of 1 and 100 ms were measured. These were similar to the time constants obtained previously by theoretical modeling.19 Due to optical chopper instability at low frequencies, the output of the second lock-in amplifier (peak heights and areas) was not used for analysis. At the lower modulation frequencies, wobble in the optical chopper contributed to sinusoidal noise patterns in the output of the second lock-in amplifier. The noise prevented use of the lower frequencies for actual detection, despite the significant advantage gained in signal intensity. Effect of Polyimide Coating on Background Signal. The preparation of the capillary for photothermal detection included removing a section of polyimide from the capillary by electric arc.20 Originally, a section slightly larger than the width of the gap was removed. It was observed, however, that a large background signal was still present when the capillary was filled with nonabsorbing solutions. The output of the first lock-in amplifier was observed using a 30-µm-inner diameter capillary with a 2-mm window. A background conductivity signal of 30.5 mV was measured when the capillary was filled with 20 mM borate buffer. When filled with 48 µM dabsyl-labeled glucosamine, a 37.5-mV signal was observed. (19) Fadgen, K. E. Doctoral Dissertation, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 2001. (20) Hoyt, A. M., Jr.; Beale, S. C.; Larmann, J. P., Jr.; Jorgenson, J. W. J. Microcolumn Sep. 1989, 1, 41-45.

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Figure 7. Pseudo-three-dimensional modeling of heat transfer using FEMLAB 3.0. (A) Model was rotated 360° around center axis to create three-dimensional environment. (B) Modeled average temperature for 4-Hz pulsing within (a) the heat region, (b) the gap, and (c) the sensing region.

The window was expanded to 1 cm and signals of 1.0 and 5.2 mV were measured for the buffer and glucosamine, respectively. For the small window, scattered light was absorbed by the polyimide, resulting in a larger than expected background conductivity signal. When the window was widened, removing all polyimide from the vicinity of the detection region, the background signal dropped. A larger percentage of the detected change was due to the absorption of light by the glucosamine, rather than by the capillary. Multiphysical Modeling of the Photothermal Event. Using Femlab 3.0 (Comsol, Inc., Burlington, MA), heat dissipation and solution flow were modeled for the conductivity cell using the heat transfer and incompressible Navier-Stokes multiphysics, respectively.21 For all models, a 50-µm-i.d., 360-µm-o.d. capillary was simulated with the heated region approximated as a 50-µm length in the middle of the gap. Simulations were run with and without electrodes present. When electrodes were present, they were simulated as 380-µm-i.d., 550-µm-o.d. cylinders with a 1.1(21) FEMLAB 2.2 Chemical Engineering Module Manual; Comsol, Inc.: Burlington, MA, 2001.

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mm gap between them. An aqueous solution was used to fill the capillary in all simulations. A 3-mm radial portion of the capillary was drawn and then rotated 360° around an axis to create a pseudo-three-dimensional entity (Figure 7A). Limitations in computing power prevented a true three-dimensional simulation from being run. The inner diameter of the capillary was defined in multiple sections: the heat region, the remainder of the gap, and the sensing region. From experimental results and from an expanded electrical conductivity model,17 it was estimated that, for a 1.1-mm gap with an excitation frequency of 100 kHz, the area of conductivity detection extended ∼1 mm underneath each electrode, for an overall sensing region of ∼3 mm. Temperatures were monitored as an average over each region. From initial simulations, it was seen that, on the time scale of the modulation (200 mW, 4 Hz), the displacement of heat due to fluid flow was much smaller in magnitude than that due to conductive heat transfer. For all subsequent simulations, the incompressible Navier-Stokes multiphysic was removed from the simulation, thus saving further on computing power requirements.

by simulation (only a 35% increase). A likely explanation for this is the difference in the way the heat was applied in each instance. For the simulation, a uniform amount of energy was applied over the entire gap. However, experimentally, the beam still maintains a Gaussian intensity pattern across the line. The majority of energy is still being input near the center of the gap. Replicating the Gaussian shape of the energy input in the simulation was not possible due to the pseudothree-dimensional construction of the model. CONCLUSION Figure 8. Comparison of measured signal for a front of 48 µM dabsyl-labeled glucosamine in 20 mM borate buffer detected using the argon ion laser focused to (A) a point and (B) a line.

It was observed that the average temperature change of the heat region was much larger than that of either the gap or sensing regions (Figure 7B). This relationship held for both modulated and nonmodulated simulations. The average temperature changes of the sensing region were ∼4.9% that of the heated region. The simulation was repeated with and without electrodes. With electrodes, a minimal disturbance of the flow of heat within the system was seen. The presence of the electrodes was not a contributing factor to the loss of detected temperature change. Experimentally, for a 200-mW, 4-Hz modulation, a 1.0-mV change in conductivity is seen on top of a 380-mV background conductivity, an 0.26% change. As solution conductivity changes 2%/°C change in temperature, this corresponds to an 0.13 °C change in temperature. Over the sensing region, the simulation predicted an 0.36 °C change for this experiment compared to a 7.4 °C change over the heated region. The experimental temperature change of the sensing region is within the same order of magnitude as that predicted by the simulation. However, if detection could occur experimentally over just the heated region, an ∼20-fold increase in signal could be achieved. The wide length of the sensing region in contrast to the small heated region leads to a decrease in the sensitivity of the detection. As a comparison, the simulation was set up with the entire gap region heated instead of just the center portion. For this simulation, the same amount of total heat was input into the system with the point focus spread over the gap region so that an identical amount of total energy would be perturbing the system. From the simulation, a 5-fold increase in signal was expected over the sensing region when the line focus was used instead of the point focus. This simulation was tested experimentally using the cylindrical lens for beam focusing in place of the microscope objective. The cylindrical lens was set within an optical rotator to allow for exact alignment of the focused line with the inner diameter of the capillary. Although an increase in signal was seen with the line focus as compared to the point source (Figure 8), the signal level was still far below what was predicted (22) Krattiger, B.; Bruno, A. E.; Widmer, H. W.; Da¨ndliker, R. Anal. Chem. 1995, 67, 124-130. (23) Wu, J.; Odake, T.; Kitamori, T.; Sawada, T. Anal. Chem. 1991, 63, 22162218. (24) Ragozina, N.; Heissler, S.; Faubel, W.; Pyell, U. Anal. Chem. 2002, 74, 44804487.

The results of the multiphysical modeling of heat transfer in the photothermal absorbance detector showed that, to detect the entire theoretical temperature rise, the detection gap should be on the order of the size of the focused light spot. Complicating this, however, is the flow of current through the conductivity cell. Using a laser light source focused to a spot, the electrodes would need to be moved extremely close to each other to fully detect the change in temperature (and subsequently conductivity). Moving the electrodes close together though does not significantly narrow the conductivity detection region. If a wide capacitive coupling distance is maintained, the effective conductivity sensing region will never be smaller than ∼2 mm. Even using a line focus to fill the gap does not enable the complete detection of the temperature change due to the wider effective conductivity sensing region. A format is needed in which the electrodes can be brought much closer together without influencing the conductivity detection. One possible format for this is microfluidics. There is, however, an inherent disadvantage to the integration of a contactless conductivity detector into a microfluidic chip. The planar geometry of the electrodes, while resulting in a decreased amount of leakage current between the electrodes, also results in a decreased capacitive coupling efficiency with the solution as compared to the coaxial geometry present in capillaries. As an alternative method, contact conductivity does not suffer from this limitation and can also be integrated into microfluidic chips. Development has begun on a microfluidic chip-based photothermal absorbance detector using a contact conductivity detection scheme. It is acknowledged that, for this detector to be a viable alternative, detection in the ultraviolet wavelength range must be accomplished. However, based on previous work on crossed-beam thermooptical detection,11,22 laser-induced capillary vibration,23 and near-field thermal lensing,24 the use of UV wavelengths does not appear to be a hindrance for the photothermal class of techniques. ACKNOWLEDGMENT This work was funded by a grant from the Waters Corporation and Grant CHE-9727505 from the National Science Foundation.

Received for review December 15, 2005. Accepted April 21, 2006. AC052223J Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

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