Anal. Chem. 1988, 6 0 , 311-316
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Propagation of Photoacoustic Waves Generated on Liquid Chromatography Columns Kathy L. Rowlen and John W. Birks*
Department of Chemistry and Cooperative Institute for Research i n Environmental Sciences (CIRES), University of Colorado, Boulder, Colorado 80309 Kenneth A. Duel1 and James P. Avery
Department of Electrical and Computer Engineering and CIRES, University of Colorado, Boulder, Colorado 80309
Photoacoustic wave propagatlon along quartz chromatographic columns is described. Three slgnlflcant wave fronts are detected In an unpacked column, two of which Involve reflections from the end flttlngs and are observed at long delay times. The least efflclent pathway In an unpacked column Is propagatlon through the solvent wlth detection occurrlng when the wave front Is dlrectiy beneath the transducer. Photoacoustic wave propagatlon on a packed microbore hlgh-perfomance llquld chromatography column occurs primarily through the solvent. The shnpllcky of the observed photoacoustic waveform on a packed column and the insensltivlty of photoacoustic technlques to scattered llght demonstrate photoacoustic spectroscopy as a useful on-column method of detectlon for packed columns.
A photoacoustic wave, in simple terms, is a sound wave resulting from a light-induced pressure fluctuation. When an excited-state molecule releases energy in the form of heat (i.e., nonradiative decay), a corresponding volume expansion occurs. In a closed system, the volume expansion results in a pressure fluctuation, or sound wave. Several authors have rigorously derived the theory of photoacoustic generation (1-5); however, very little detailed work concerning the propagation of photoacoustic waves has been conducted. In this paper we present the results of a study designed to elucidate photoacoustic wave propagation along the length of quartz chromatographic columns. In 1982, Lai et al. (6) employed photoacoustic spectroscopy (PAS) as a detection method for high-performance liquid chromatography (HF’LC). Although Oda and Sawada (7) were the first to apply PAS to HPLC, Lai et al. developed a unique and simple detection cell that could be easily fitted at the exit of a column. The cell was constructed of a quartz “column” 11 cm in length with a 1-mm i.d. and a 4-mm 0.d. A piezoelectric transducer, attached to the quartz detection column via an aluminum coupling cylinder, served as the detector. The aluminum cylinder (6.4 mm in length) was attached tightly around the outer diameter of the quartz column. A nitrogen laser, focused at a specific point on the quartz, was used to excite analytes as they flowed through the cell. Detection limits similar to, or better than, UV absorbance detection limits were reported for a variety of compounds. By comparing calculated acoustic velocity (distance the sound wave traveled divided by the elapsed time between excitation and arrival time at the detector) with known acoustic velocities, the authors were able to conclude that the photoacoustic wave traveled primarily through the solvent rather than transferring to the quartz. This result was further substantiated by the observation that an air bubble within the solvent completely extinguished the photoacoustic signal.
Later, in 1983, Lai et al. (8) improved the design of the quartz detection column by attaching the piezoelectric transducer to the end of the quartz such that an acoustic wave traveling in the solvent would directly impinge on the aluminum coupling cylinder. The use of three transducers along the cell, two attached in the “end-on” arrangement and one attached to the outer diameter, allowed the authors to compare the relative responses. They found the end-on attachment to be approximately a factor of 10 more sensitive. In addition, they observed secondary waves at one of the end-on transducers. The authors noted that the transducer did not detect primary waves but that it picked up acoustic waves that appeared to be of “secondary” origin, since the arrival times were longer than those predicted and the waveform more diffuse. The authors used the term “secondary” to imply a mode of propagation other than direct travel to the transducer. The nature of these secondary waves, their mode of propagation, remained unclear. We demonstrate here the use of signal averaging over extended time intervals and selective acoustic damping to further characterize photoacoustic propagation within a quartz detection column similar to the one employed by Lai et al. (7, 8). By sampling the transducer output for sufficiently long periods after excitation, all acoustic wave fronts can be observed. Delay time analysis, i.e. comparison of calculated acoustic velocities with known acoustic velocities (literature values), is used to predict the most likely pathway each photoacoustic front traveled to reach the transducer. Selective positioning of an acoustic insulator, in this case an air bubble, provides the means to verify or reject the proposed pathway. In addition, and perhaps more importantly, we extend these techniques to the evaluation of a photoacoustic wave generated on a packed chromatographic column to demonstrate the use of photoacoustic detection as an on-column detection method for microbore HPLC. We are currently developing photoacoustic spectroscopy as a potential means of implementing what we described earlier as “whole column detection chromatography” (9).
EXPERIMENTAL SECTION A block diagram of the instrumental arrangement is shown in Figure 1. A CMX-4 tunable dye laser, with Rhodamine 590 dye, was employed as the excitation source. The diameter of the unfocused beam at the point of intercept on the quartz column was -3 mm. Beam power was measured with a pyroelectric detector (Scientech, 360001). The chromatographiccolumns were constructed from 1-mm i.d. X 6-mm0.d. quartz (Quartz Scientific) and were 19.3 and 26.7 cm in length for the unpacked and packed columns, respectively. The ends of the quartz columns were polished with silicon carbide to ensure a flat surface. Teflon ferrules were used to seat Swagelok zero dead volume end fittings. Kel-F encased, 1-mm-diameter,stainless steel frits were used in order to reduce the chance of fracturing the quartz while adjusting the end fittings. In this system, the Teflon ferrules held column
0003-2700/88/0360-0311$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60,NO. 4, FEBRUARY 15, 1988
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Figure 1. Block diagram of photoacoustic on-column detection instrument. PM and INJ represent power meter and injector, respectively.
pressures of up to only -400 psi; therefore, larger particle sizes were used for the stationary phase. The columns were handpacked with 40-pm ODS (Alltech Associates). The pressure drop across a 20-cm column (0.1 mL/min) was typically 200 psi. The efficiency of each column was determined with a uracil/naphthalene/anthracene mixture by using UV absorbance postcolumn detection (Kratos, Spetroflow 773). The number of theoretical plates per meter was in the range of 800-1000. Poole and Schutte (IO)have recommended 1000 plates/m as the minimum acceptable efficiency for 40-pm reverse-phase packing. The column, injector (Rheodyne 7410, 1 p L sample loop), and preamplifier were all mounted on a calibrated translation stage in a fixed configuration, with flexible Teflon tubing from the pump (Waters SOOOA) to the injector. This arrangement allowed discrete positions along the length of the column to be illuminated by manually adjusting the translation stage. A cylindrical piezoelectric transducer (Vernitron, PZT 5A, 8-8031) was employed as the detector and was attached directly to the quartz with a cyanoacrylic adhesive. The electrical leads were carefully soldered to the transducer using Ni/Sn solder. These piezoelectric cylinders are designed to respond to changes in circumference as well as changes in length. In order to determine which direction of change was more sensitive, a function generator (HP 8111A) was attached to one transducer and used as an acoustic transmitter. Another transducer, the one to be tested, was positioned as a receiver. Monitoring the amplitude output of the receiver on an oscilliscope as the frequency of the transmitter was scanned resulted in an amplitude versus frequency spectrum. In general, the transducers were found to be more responsive to changes in length. Therefore, for subsequent experiments the electrical leads were placed on opposite ends of the transducer to detect changes in length. To minimize acoustic pickup, the translation stage and pump were enclosed in an acoustic box constructed of lI4-in. plywood. The box was lined with urethane foam and/or '/*-in. aluminum and mounted on '/2-in.foam rubber padding. In addition,the stage rested on 1/4-in. Styrofoam and a 'I4-in. vinyl mat. Masking for the piezoelectric transducer consisted of a simple aluminum "blinder" positioned between the transducer and the laser beam/column intercept point. This blinder served to prevent scattered light from reaching the transducer. The preamplifier, designed and constructed in our laboratory, was operated at a gain of 2000 with a frequency range of 800 Hz to 200 kHz (-3 dB). The preamplifier was powered by a 15-V dc source and positioned as close as possible to the transducer in order to reduce noise pickup. After amplification the signal was converted from analog to digital (nine-bit resolution) and signal averaged (Nicolet, 1170). An averaging rate of 1psladdress proved to be sufficient to prevent aliasing. The 4K memory capacity of the averager was split into 4 subgroups, so that 1024 addresses (or 1024 1s) were available for each signal. The signal event (delay time + ringing) was observed to significantly decay within 500 gs. Typically, 16-128 pulses were averaged. Triggering was provided directly from the laser (Sync Out), and timing accuracy of the laser trigger was confirmed by comparison with a photodiode trigger. An oscilliscope (HP 1726A, 275 MHz) was
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Flgure 2. Photoacoustic signal as excitation point is moved farther from the transducer. For a, b, c, and d the distances between the excitation point and the transducer are 4 , 6 , 8, and 10 cm, respectively. The analyte is 2.44 X 10" M Cr(OCH,)TPP in methanol. The column is unpacked. Other conditions: flow rate is 0.1 mL/min.; 2.2 mJ/pulse; 595-nm light; 5 Hz repetition rate with 32 pulses averaged.
used to simultaneously monitor the amplified signal and the trigger. Upon completion of averaging, the digitized waveform was transferred serially to an IBM XT via a RS 232 port. Due to electronic limitations, each memory subgroup transfer to the computer required manual initiation. The process of data aquisition was therefore limited by the rate at which each memory subgroup could be transferred and reset. Since the time required for transfer to the computer was on the order of 30 s, it was impossible to adequately sample a chromatographic peak on the column with this system. To remedy this situation, a transient digitizer with continuous and real time transfer is currently being developed in our laboratories. Because of the limitations mentioned above, the photoacoustic signal as a function of concentration was determined by allowing the entire column to equilibrate with the analyte solution. Background signal was established as the acoustic response with only solvent on the packed column. Between each subsequent analyte concentration solution, the column was "cleaned" with solvent and the background redetermined. A porphyrin (Cr(0CH,)TPP) was chosen as the model compound due to its extinction coefficient at 595 nm (-9700 L/(mol cm)) and its nonradiative quantum efficiency (-1). Methanol was used exclusively as the solvent in order to simplify acoustic characterization. The solutions were prepared by 1:2 sequential dilutions of a moderately concentrated standard. Absorptivity values (a)were determined with a Hewlett-Packard 8451A diode array spectrometer.
RESULTS AND DISCUSSION Unpacked Column. Wave Front A. Figure 2 shows a sequence of photoacoustic signals generated a t various distances from the transducer. With the excitation point 4 cm from the transducer (Figure 2a), there are a t least three (A, B, C) distinguishable wave fronts. Delay time is defined as the elasped time from excitation to the first positive excursion
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
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Figure 3. Acoustic propagation in solvent. To the left of the figure, the angles a and (3 represent the angles of refraction into the quartz
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for transverse and compressional waves, respectively. Two waves result from a single incident wave at the interface. To the right of the flgure directional arrows show the transfer of the photoacoustic wave from the methanol through the quartz to the transducer at normal incidence.
closely space arrows shown at the left end frit indicate an interaction, which allows the wave front to transfer from the solvent to the quartz. Wave front C is represented by the directional arrows so labeled. Front C travels to the right through the sovlent from the point of origin. Front C is then reflected at the right end of the column and travels back to the left end within the solvent. At the left end, C undergoes a similar transfer process from the solvent to the quartz.
of the wave. A plot of the distance traveled by wave front A, assuming the front traveled directly to the transducer (i.e. 4, 6, 8, or 10 cm), versus its measured delay time results in a slope of (1.0 f 0.1) x lo5 cm/s. On the basis of agreement between the measured acoustic velocity and the literature value of 1.12 X lo5 cm/s (II),we infer that wave front A arrived at the transducer via transport through the methanol. This result is commensurate with the findings of Lai et al. (6). Although Lai et al. (6) concluded that a photoacoustic wave generated within a quartz cohmn traveled primarily in the solvent, a clear explantion of how the wave was detected by a transducer attached to the outer diameter of the quartz was not given. In order to understand how photoacoustic waves propagate, it is important to consider parameters which are known to govern physically generated acoustic waves. The efficiency of acoustic transfer across a boundary is described by the ratio of intensity transmitted to incident intensity 2p‘c ‘
cos p’ p‘c ’ -+cos p
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which reduces to
at normal incidence. Here 2 is the acoustic impedence at 90” and is analogous to refractive index for electromagnetic waves, p is density, c is acoustic velocity, y is the angle of incidence, and (3 is the angle of refraction (see Figure 3). The primed values are those of quartz, and the unprimed values are for methanol. Note that for a quartz/methanol boundary, at normal incidence, the transmission coefficient is 0.22. The laws of both reflection and refraction hold for acoustic waves; therefore, a t angular incidence the transmission can be sigincidence results nificantly less than 22%. In addition, an* in a phenomenon known as mode conversion at the interface between a liquid and a solid (12). Mode conversion may be simply described as the creation of additional acoustic waves from a single incident wave. For example, a compressional wave incident on the surface of a solid at angles less than 90” will produce two waves within the solid, a compressional wave and a transverse wave, each of which travels at a unique velocity. A compressional wave is one that oscillates in the direction of propagation. A transverse wave oscillates perpendicular to the direction of propagation. The acoustic velocities for these types of waves are well-known for many solids (11);the values for quartz are 5.90 X lo5 and 3.75 X lo5 cm/s for compressional and transverse waves, respectively.
e 4. The bold directional arrows In this figure represent wave front B which travels through the solvent from the point of origin. The small,
Consideration of the results described above leads us to conclude that the signal detected at an acoustic velocity corresponding to that of methanol must reach the transducer in the manner shown in Figure 3. Due to the large impedence difference between methanol and quartz (-10) and mode conversion at the interface, it is unlikely that sufficient energy could be transferred at angles much less than 90”. Therefore the detector does not “see” the photoacoustic wave until the front is almost directly beneath the transducer. At that point, as shown by the directional vectors on the right side of Figure 3, a component incident at the interface travels directly through the quartz walls to interact with the transducer. The wave most likely t
