LITERATURE C I T E D (1) A . C. Moffat and E. C. Horning, Biochim. ~;ophys.Acta, 222, 248 (1970). (2) A. C. Moffat and E. C. Horning, Anal. Lett., 3, 205 (1970). (3) S. B. Matin and M. Rowland, J. Pharm. Sci., 61, 1235 (1972). (4) A. C. Moffat, E. C. Horning, S. B. Matin, and M. Rowland, J, Chromatogr., 66, 255 (1972). (5) A. R. Mosier, C. E. Andre, and F. G. Viets, Jr., Environ. Sc;. Techno/., 7, 642 (1973). (6) R. E. Weston and B. 6. Wheals, Analyst(London), 95, 680 (1970). (7) E. W. Day, Jr., T. Golab, and J. R. Koons, Anal. Chem., 38, 1053 (1966). (8)S. Kawai and 2 . Tamura, Chem. Pharm. Bull. (Tokyo),16, 409 (1971).
(9) K. imai, M. Sugiura, and Z. Tarnura, Chem. Pharm. Bull. (Tokyo),19,409 (1971). (10) J. p. Chaytor, B. Crathorne, and M. J. Saxby, J, Chromatogr., 7 0 , 141 (1972). (11) L. J. Papaand L. P. Turner, J. Chromatogr. Sci., I O , 744 (1972). (12) Y. Baba, Bull. Chem. Soc. Jpn., 48, 270 (1975). (13) Y. Hoshika, J. Chromatogr., 115, 596 (1975). (14) Y. Hoshika and Y. Takata, J. Chromatogr., 120, 379 (1976). (15) ‘f. Hoshika, Anal. Chem., 48, 1716 (1976).
RECEIVEDfor review August 27, 1976. Accepted December 27, 1976.
Multiple Ion Detection System with Miniscan Facilities and Expanded Mass Range for Magnetic Sector Mass Spectrometers Francisco Artigas’ and Emilio Gelpi’” lnstituto de Biologia Fundamental, Universidad Autonoma de Barcelona, Avda. S. Antonio M. Claret, 171, Barcelona, 12 Spain
Manuel Prudencio,2 J. Antonio A10nso,2 and Juan Baillart Perkin-Elmer Hispania S.A., Barcelona, Spain
A new four-channel multiple ion detection (MID) system is described. By adding a A V of up to 1100 volts to the nominal accelerating voltage ( V ) , a mass range of about 30% ( V = 3600 volts) to 90% ( V = 1200 volts) can be covered, thus providing a remarkably wide range compared to other MID units. The system incorporatesa ramp voltage, which added to V allows a small scan around each one of the peaks focused, as well as holding amplifiers in each channel. These features combined with the excellent stability of the Ionization chamber against voltage changes mlnlmlre the errors due to defocusing effects, permitting the addition of voltages as high as those described. The scan time is continuously adjustable between 0.5 and 12 s and the swltching time between two adjacent channels is 50 ms. The circuit design, its operation, and some aspects of the performance of this unit are described.
The advantages of using a MID unit coupled with GC-MS for selected ion monitoring have been pointed out by several authors. Since the initial contributions in this field ( I , 2), a great number of papers reporting the use of a MID accessory have been published ( 3 , 4 ) .Likewise, the use of this technique in the analysis of biological samples has been steadily growing because of its great potential in terms of sensitivity and specificity ( 5 ) . We have recently attempted to use a single focusing mass spectrometer equipped with standard peak matcher facilities for selected ion monitoring (6, 7 ) . However, this implies multiple GC injections to be able to monitor various ions. Also, the use of deuterated internal standards for accurate quantitative measurements requires monitoring more than one ion. Thus, in order to analyze the different components of any given sample in a reasonable time, it is important to have the capability of focusing several ions simultaneously. Although this capability is provided by the standard MID systems del Present address, Instituto de Biofisica y Neurobiologia, Barcelona, Spain. Present address, TBcnicas ABM, Barcelona, Spain.
C.S.I.C.,
scribed for magnetic instruments, the effective mass range covered in this way is relatively limited by variations in ion focusing a t different accelerating voltages (8, 9). As the effective mass range depends upon the magnitude of the AV, values added or more commonly substracted (voltage attenuation), from the nominal accelerating voltage ( V ) ,these values should be as high as possible without introducing unstabilities in the ionization chamber of the mass spectrometer. Using the device described here, we are able to add AVLvalues, from 0 to 1100 volts, to the nominal accelerating voltage in each one of the four channels used without any significant disturbance in the performance of the ionization chamber. Also, the stepping up of V means that sensitivity is not reduced at the high masses being actually increased at the lower masses in contrast to other systems ( 5 ) . After establishing a method for using the peak-matcher of a Hitachi-Perkin-Elmer mass spectrometer as a device to monitor only one ion per injection (6) and improving i t by rapid changes of the magnetic field during the time intervals between the elution of the components of interest so that two or three masses can be monitored per injection ( 7 ) ,we still faced the need for a better capability in the sense described above. Since there is no MID unit available on the market to be coupled with this equipment, the development and construction of a simple and versatile MID accessory seemed to be necessary. This MID unit must overcome several problems, like the absence of mass marker and computer as well as the excessive “drift” of the mass peak being focused, due to the instability of the magnetic field of the mass spectrometer. On the other hand the stability of the magnetic field improves significantly if the magnet is allowed to equilibrate thermally for several hours before a run. However the stability thus achieved is lost when widely differing m/e values need to be focused upon from run to run. The focusing effect thus produced by the variations of the magnetic field usually requires the injection of a standard either to confirm or adjust the focus on the selected masses. Lately Klein et al. (8)have used a feedback circuit with a Hall probe to stabilize the magnetic field and Holland et al. (9) have described a computer autofocus system for continuous fine adjustment of optimal focusing. However in the absence ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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A Figure 1. Block diaaram of the MID-MS unit I.S. = ion source, M = magnet, E.M. = electron multiplier,O.R.= oscillographic recorder, P.R. = pen recorder. Parts under the dotted line illustrate the various components added to the standard MS system. l A , lB, and 1c show the circuit diagrams Of the programmed power supply, digital programmer, and holding amplifiers, respectively. Resistance and capacitance values are given in k f l and pF, respectively
of this kind of sophisticated circuitry and computer facilities, this problem must be approached in a different way. To avoid any major modifications and/or additions to our standard GC-MS unit, either mechanical or electronic, and especially those of a permanent nature, we planned on a system built around the peak matcher unit and completely independent of the rest of the electronic circuits of the ion source, magnet, and electron multiplier.
EXPERIMENTAL Instrumentation. A Perkin-Elmer 3920 gas chromatograph was used, combined through a single stage gold jet separator with a Hitachi-Perkin Elmer RMU-6H mass spectrometer, equipped with a peak matcher model MK-14B and a glass inlet system. Two dual pen recorders (Perkin-Elmer 56) were used to monitor the four channels. A schematic diagram of the MID accessory as well as circuit diagrams of the programmed ion source, digital programmer, and holding amplifiers are shown in Figure 1,A, B, and C, respectively. All the circuits have been designed and built by the authors using standard electronic components. Derivatization Procedures. The compounds used to test the setting up of the MID unit were: PFK, tryptophan (TP),tryptamine (T), indoleacetic acid (IAA), serotonin (5-HT), and 5-hydroxyin544
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doleacetic acid (5-HIAA). Except for PFK, they were derivatized according to described procedures ( 6 , l O ) . The carboxyl groups are methylated (Me) and the primary and secondary amine groups are acylated to the corresponding mono-, bis-, or tris-pentafluoro propionyl derivatives (abbreviated as lPFP, PPFP, or BPFP, respectively). The chromatographic conditions are shown in the footnotes of the figures.
PRINCIPLE AND DESCRIPTION OF THE MID UNIT The relationship mle = KH2/V shows that, by fixing the magnetic field of the mass spectrometer, it should be possible to focus the top of any given mass peak. So, by means of an accelerating voltage alternator, as described Sweeley et al. ( I ) , it is possible to collect simultaneously the signals from three or more values of mle. After the initial work, several modifications were introduced ( 2 , lI ) , giving rise to more sophisticated analogic MID systems. Taking our technical capabilities and specifications into account we have developed a MID system consisting of (Figure 1): A) a programmed 0-1100 volts voltage source, B) programmer, C) variable ramp generator, D) signal distributor, E) four holding amplifiers, F) oscilloscopic monitor.
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The additional voltage source A, which is connected to the standard power supply of the RMU-GH, adds to the nominal accelerating voltage in each one of the four monitoring channels a regulated voltage increment (AV), varying continuously from 0 to 1100 volts. However, as the magnetic field of the mass spectrometer is not as stable as the specifications of a fixed voltage MID would require, we thought of using a small adjustable voltage scan around the peak being focused (8,9,12,13). Figure 2 shows the shape of the function voltage vs. time. The voltage sweep (V,) acts equally on each channel during time t ( t continuously adjustable between 0.125 and 3 s). This voltage is provided by the ramp generator (C) of the peak matching unit and its value is selected according to the number of peaks desired to be focused in each channel. Thus each one of the four channels receives a regulated voltage increment given as a function of time A V ( t )which is added to the nominal accelerating voltage ( V ) .This A V ( t )is the sum of a fixed AV supplied by the programmed power supply (A) and the variable voltage sawtooth wave (V,) supplied by the
ramp generator (C), thus obtaining effectively a “miniscan” around each of the selected masses. The programmer B provides the time sequence for each channel so that the programmed voltage is applied to the ion source and a t the same time the output signal is sent to the corresponding holding amplifier. In order to get this time sequence, the sawtooth wave produced by the ramp generator C is compared to fixed preselected voltages by means of four comparators so that only one quarter of the full ramp amplitude is used in every channel, thus allocating a quarter of the total scanning time ( 4 t , see Figure 2 ) for every channel. This sawtooth wave is also applied to the horizontal amplifier of the monitor F, thus synchronizing the voltage source A and the signal distributor D with the horizontal scan of the monitor. The amplifiers are connected to the detector only during the time that the ions are being focused, but not during the switching time, to overcome the possibility of spikes due to transients (14). In relation to the signal/noise ratio, it is important to have the capability of selecting the scan times. A rapid scan is used ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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when selecting the mass peaks, while a slower scan is used for measuring, so that potentiometric recorders can be used with the system. The signal distributor D directs the signal coming from the electron multiplier of the mass spectrometer to each holding amplifier. Due to the fact that the signal distributor is driven by the programmer, each one of the recorder pens collects only the signal arising from the channel to which it has been connected, maintaining the peak value until the programmer returns to the same channel, which then sends the pen to a level higher or lower on the chart paper depending on the ion abundance at that moment. The signal thus obtained when a peak elutes from the gas chromatograph is registered in a discontinuous stepwise fashion. The amplitude of the steps depends on the scan time, so that rapid scans produce smoother eluting profiles. Each channel has a holding amplifier built with a peak detector and a S/H amplifier. During the recording of the chromatographic peak, the holding amplifiers eliminate the oscillations due to the return of the signal to baseline in every scan, thus providing a more linear response when pen recorders are used. We had noticed previously that, using the peak matcher as a specific detector for one m / e value (€4,in a similar way as described by Boulton et al. (151, the response was more linear using UV recorders than potentiometric recorders because of the relatively high full scale response time of the latter (0.5 s). With holding amplifiers, this problem is overcome by elimination of the scan oscillation (13)as can be seen in the left-hand side of Figure 3. 546
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Figure 3. R Chromatographic signal obtained at the output of the MID system. B: Signal corresponding to the same peak at the output of Pen Recorder (see Figure 1). The width of the each step in A represents the total scan time (4t, see Figure 2). Paper speed = 8 cm/rnin
The system is completed by filter (low pass filters and reject filter, tuned to 50 Hz to eliminate residual hum), zero, and bucking (background substraction) controls as well as two independent gain controls: one of these acting upon the four channels at a time, the other varying the gain of each channel, independently of the others. The latter is used to compensate for the differences in sensitivity due to the different accelerating voltages acting in every channel.
FOCUSING OPERATIONS The desired masses are selected and focused as follows: with a five-position selector knob, it is possible to switch on each one of the four preset AVLvalues (corresponding to positions 1 to 4 of the selector knob). That is, when the selector is in position 1 the region of the mass spectrum around the peak of interest appears on the screen of the oscilloscopic monitor F. By decreasing the amplitude of the sawtooth voltage wave (V,.),this region can be expanded from the left of the screen so that the peak of interest takes only a quarter of the total screen width. This procedure is repeated for the other positions of the selector (that is, when channels 2,3, or 4 are on), thus focusing the four peaks. In position 5, the programmed voltage source changes sequentially the four AV values added to the nominal accelerating voltage, thus monitoring successively each one of the four preselected peaks. At this “Auto” position, the oscilloscope screen is divided into four independent “windows”. Figure 4 shows four peaks, each appearing in its corresponding “window”. These windows are separated by a programmer driven upward spike (the white dots over the baseline in Figure 4). These light frames are helpful in order to avoid confusion between peaks of adjacent channels, corresponding
P F K MASS S P E C T R U M AT 4700 V
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Figure 4. Real time display of four peaks on the oscilloscopic screen The channel width ("window") corresponds to the distance between two of the light points over the baseline
their width to the switching time between channels (about 50 ms). Also if the operator selects a relatively large ramping voltage V,, other adjacent ions can be monitored andlor recorded by the same channel if needed. The operator focuses the selected ions by means of a reference compound such as PFK or more conveniently the stationary phase background. In our experience (6, 7,16) the latter provides a very simple to use and reliable quantitative mass marker which we use in routine operation of the system. For instance, mle 281 from an SE-30 or OV-17 is helpful to locate mle 276 and 289, both corresponding to perfluoroacylated indolic compounds while mle 429, from the same phases allows the correct focusing of mle 438, given by perfluoroacylated 5-hydroxindolic compounds. If in doubt an adequate internal standard is run to verify the correct focusing of each mass peak.
RESULTS AND DISCUSSION Figure 5 shows the mass spectrum of low boiling PFK obtained by scanning the magnetic field at an accelerating voltage of 4700 volts. The left-hand side inset shows a GCMID profile in which IAA-Me-1PFP and T-2PFP are present. This profile has been obtained by focusing mle 129. Figure 6 shows the relationship between the mass peak height and the AV added over 3600 volts. A constant leak of low-boiling PFK was introduced into the ion source through a molecular leak and the peak height of mle 69 was measured at different values of the AV added. The ion optics (lens and repellers) were adjusted for maximum sensitivity at 4700 volts at the beginning of the measurements. Although these results indicate that the performance of the ionization chamber is not affected by high voltage changes, we have also tested the degree of defocusing due to high voltage switching by comparing data obtained at two different conditions: i) Absence of voltage changes in the ion source, that is, data obtained with the same voltage acting in every channel, ii) High voltage changes in the ion source. For this purpose, we have compared the ratio between the intensities of two mle values of PFK (mle 100 and 119) at these two conditions, according to the following procedure: i) Both masses were registered on one channel at the same fixed accelerating voltage (4700 volts). In this case the experimentally determined peak height ( H ) ratio without voltage switching is equal to H mle 119 (at 4700 volts) = 1.98 H mle 100 (at 4700 volts)
Figure 5. PFK mass spectrum obtained by scanning the magnetic
field MS-MID conditions: Vnornina,= 3600 volts. A V = 1100 volts. Electron energy = 70 eV. Emission = 80 PA. Target = 40 PA. Analyzer pressure = 1. mm Hg. Ion source temperature = 200 OC. Inset; GC profile of IAA-Me-IPFP and T-2PFP at m/e 129. GC conditions: glass column 2.5 m X 3 mm i.d., packed with 3 % SE-30 on Gas Chrom 0 100/120 mesh. Column temperature = 205 OC. Flash heater and jet separator were set at 250 OC. MS-MID conditions: V,,., = 3600 volts A V = 1100 volts. Analyzer tube pressure = 3 X mm Hg. Other conditions were the same as for PFK spectrum
ii) Likewise the same ions were also registered on two different MID channels, operating a t 3830 and 4700 volts, respectively. The ratio obtained with accelerating voltage switching was
H m/e 119 (at 3820 volts) = o.29 H mle 100 (at 4700 volts) This value should be corrected for equivalent sensitivities. From the sensitivity vs. AV data of Figure 6, also obtained a t fixed accelerating voltage, it can be shown that: response a t 4700 volts = 7.18 response a t 3830 volts Thus 0.29 X 7.18 = 2.08, which allows a direct comparison of the two experimental derived ratios a t fixed and alternating accelerating voltages. The difference of about 5% proves that the operation with voltage changes as high as 870 volts does not introduce any significant degree of defocusing or ion source instabilities. Figure 7 shows the response of the MID unit for an injection of IAA-Me-lPFP, T-BPFP, TP-Me-2PFP, 5-HIAA-MeBPFP, and 5HT-3PFP (about 5 ng of free acid, amine, and aminoacid), obtained by monitoring characteristic masses of these compounds (Le., 276 for indoles and 438 for 5-hydroxyindoles). The analysis of the amine is verified by focusing on mle 289 and mle 451, corresponding to T-2PFP and 5HT3PFP, respectively (6).I t is important to point out that with low nominal accelerating voltages, masses widely separated can be focused simultaneously. As can be seen in Figure 7, a t a nominal accelerating voltage of 1200 volts it is possible to cover a wide mass range; from 276 to 451, without the need to ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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Figure 7. Four-channel recording of TP metabolites (about 5 ng of free acid, amine, and aminoacid) at m/e = 276, 289, 438, and 451 Figure 6. Sensitivity values obtained by focusing m/e 69 from a constant leak of PFK at different values of A V over 3600 volts MS-MID conditions: VnOmina,= 3600 volts. Emission = 80 @A.Target = 40 @A. Electron energy = 70 eV. Ion source temp. = 200 O C . Source slit = 0.2 mm. Collector slit = 0.1 mm
add the maximum A V provided by the system. This means that this particular metabolic pathway of TP can be analyzed with only one injection. The mass range covered by this MID unit is from about 30% at an accelerating voltage of 3600 volts to about 90% a t 1200 volts. The four peaks are monitored by two dual pen potentiometric recorders, and it can be seen that the shape of the peaks is approximately equivalent to those obtained with a FID or TIM detector. The electric noise produced by this MID unit is approximately the same as that existing before input of the signal to MID circuitry (i.e., a t the output of pen recorder (P.R. in Figure 1)).This means that neither the "holder" amplifiers nor the switching between channels produces a significant degree of additional noise. On the other hand, the use of low scan times is compatible with high time constant filters, which leads to a decrease in noise and therefore increase in sensitivity, expressed as S/N ratio. This device is being currently applied successfully in our laboratory to the analysis of tryptamines, catecholamines, prostaglandins ( 1 6 )and drug metabolites (17) in biological samples, using deuterated compounds as internal standards. Amounts of only 10 pg of 5-MTOL (5-Methoxytryptophol) can be easily detected with this system (18). It must be emphasized that the addition of a voltage ramp to the accelerating voltage affords a good solution to the problems common to the use of magnetic sector instruments in the MID mode: (8,9,12) e.g., losses of sensitivity due to the defocusing of the mass peak of interest and limitation in the mass range covered. Obviously, in this way, the expansion of mass range achieved by addition of a AV of up to 1100 volts does not represent a critical step in relation to the exact fo548
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
GC conditions: gx. column (2 m X 2 mm 1.d.) packed with 5% SE-30 on Gas Chrom Q 100/120 at 205 OC. Jet separator = 240 O C . Injector = 240 O C . MS-MID conditions: V,,,, = 1200 volts. Emission = 80 PA. Target = 40 PA. Electron energy = 70 eV. Analyzer pressure = 2.5 mrn Hg. Scan time = 1.25 s (0.31 s. per channel). Ion source temperature = 200 OC
cusing of any mas peak, as opposed to fixed channel voltage MID units. The slight m/e drifts due to the variations of the magnetic and/or electric fields can be monitored continuously on the oscilloscope screen, thus allowing a real time control of peak focusing during the analysis which can be corrected either by manually adjusting the magnetic or electric fields. This means that any errors due to an incorrect measurement of the ion abundance are reduced to a minimum. In summary, a n inexpensive MID accessory has been developed in order to overcome the limitations posed by the MS instrumentation available in the laboratory, especially in regards to the drift of mass values, the mass range covered, and the recording system. Also, as illustrated on Figure 1, the system is an accessory in the sense that all of its various components are external to the basic mass spectrometer which has not undergone any modification as shown above the dotted line of Figure 1.
LITERATURE CITED (1) C. C. Sweeley, W. H. Elliot, I. Fries, and R. Ryhage, Anal. Chem., 38, 1549 (1966). (2) C. G. Hammar, B. Holmstedt, and R. Ryhage, Anal. Biochern., 25, 532 (1968). (3) E. Costa and E. Holmstedt, Ed., "Gas Chromatography Mass Spectrometry in Neurobiology", Advances in Biochemical Psychopharmacology,Vol 7, Raven Press, New York, 1973. (4) L. Palmer and B. Holmstedt, Sci. Tools, 22, 1215 (1975). (5) F. C . Falkner, B. J. Sweetman, and J. T. Watson, Appl. Specfrosc. Rev., 10, 51 (1975). (6) J. Segura, F. Artigas, E. Martinez, and E. Gelpi, Biomed. Mass Spectrom., 3, 91 (1976). (7) E. Peraltaand E. Gelpi, Clin. Chirn. Acta, 73, 13-18 (1976). (8)P. D. Klein, J. R. Hauman, and W. J. Eisler, Anal. Chem., 44, 49 (1972): (9) J. F. Holland, C. C. Sweeley, R. H. Thrush, R. E. Teets, and M. A. Bieber, Anal. Chem., 45,308 (1973).
(10) E. Gelpi, E., Peralta, and J. Segura, J. Chromatogr. Sci., 12, 701 (1974). (11) C-G. Hammar, and R. Hessling, Anal. Chem., 43, 298 (1971). (12) W. F. Holmes, W. H. Holland, B. L. Shore, D. M. Bier, and W. R. Sherman, Anal. Chem., 45, 2063 (1973). (13) L. D. Gruenke, J. C. Craig, and D. M. Bier, 23rd Annual Conference on Mass Spectrometry and Allied Topics. Houston, Texas, May 25-30, 1975. (14) R. W. Kelly, J. Chromatogr., 71, 337-339 (1972). (15) A. A. Boulton, S. R. Philips, and D. A. Durden, J. Chromatogr., 82, 137 (1973). (16) J. Rosello, J. Tusell, and E. Gelpi, J. Chromatogr.. in press.
(17) M. C. Sdnchez, J. Colome, and E. Gelpi, J. Chromatogr., 126, 601 (1976). (18) C. Sutiol and E. Gelpi, unpublished results.
RECEIVEDfor review March 11, 1976. Accepted December 9,1976. The authors thank the Comision Asesora de Investigaci6n Cientifica y TBcnica for Research Grant 100.01.721/ 74.
Opto-Acoustic Trace Analysis in Liquids with the FrequencyModulated Beam of an Argon Ion Laser W. Lahmann,* H. J. Ludewig, and H. Welling lnstitut fur Angewandte Physik, Technische Universitat, Hannover, Welfengarten 1, Federal Republic of Germany
The opto-acoustic determination of low absorptlons has been appiled to the assay of small amounts of nonfluorescent absorbers In liquids. The output of an argon ion laser at 488 and 514 nm was used for an excitation of the sample at a perlodicaily alternating wavelength. This dual-wavelength operation enabled a considerable reduction of the background signal. A detection limit of 9 X 1O’O molecules per cm3 (12 ppt) has been achieved.
Lasers have proven to be a powerful analytical tool, e.g., in atomic fluorescence flame spectroscopy by replacing the hollow cathode or electrodeless discharge lamp excitation by a nitrogen-laser-pumped dye laser, as reported by Omenetto et al. (1). The most dramatic improvement in detection sensitivity by use of lasers was attained in non-emitting homogenous gas media. The highest sensitivity reported so far has been achieved by Fairbank, Hansch, and Schawlow (2) with a CW dye laser resulting in a detection limit of only 100 Na atoms per cm3. Despite these outstanding achievements in the gas phase, laser fluorimetry in condensed media may turn out to be an even more important analytical application of fluorescence as most analytical problems involve matter in the condensed phase. Zare has given a review of the corresponding experiments performed so far ( 3 ) ,e.g., concerning a combination of laser fluorescence detection with chromatographic separation. In his review a detection limit of 7.5 X lo8 molecules of Rhodamine 6G per cm3, corresponding to 0.0006 ng per cm3 is reported. However, in trace analysis the analytical procedure quite often has to apply a nonfluorescent, highly absorbing indicator for the substance to be detected. The concentration of this indicator is usually determined by absorption spectrometry with conventional spectrophotometers. Here the use of lasers, as for fluorimetry, should allow a considerable improvement in the detection limit, as it is possible t o measure substantially lower absorptions by use of lasers in conjunction with optoacoustic absorption spectrometry. In opto-acoustic absorption measurements a periodically interrupted laser beam is directed into a sample cell. The absorbed radiation is converted into heat via nonradiative processes thus producing periodic pressure fluctuations which are detected with a sensitive microphone. This opto-acoustic determination has been successfully applied to the detection
of ultralow gas concentrations; a comprehensive review is given by Dewey ( 4 ) . So far the opto-acoustic trace analysis has been confined to the gas phase. This article describes the opto-acoustic method utilized for liquid media. This method may find widespread application because of the ubiquity of liquids in trace analysis. In contrast to opto-acoustic absorption measurements in the gas phase, the background absorption of the chief constituent (solvent via air) is considerable, producing a substantial opto-acoustic signal. For an elimination of this background a dual-wavelength excitation with a laser beam is applied where the absorption by the solvent is equal for both exciting wavelengths, but highly different for the dissolved substance to be detected.
EXPERIMENTAL Figure 1 shows the experimental setup. The output beam of an argon ion laser operating in the “all lines mode” is separated by the prism P1 into its spectral components. The perforated plate S transmits the two most powerful beams at 488 and 514 nm and screens off all the other beams of the argon ion laser. (In Figure 1 these beams are already omitted for lucidity and the angle between the two remaining beams at 488 and 514.4 nm is somewhat exaggerated for the graphic representation). The two beams are spatially reunited with the lens L and the second prism P2, and the recombined beams are directed via mirror M into the sample cell. The chopping wheel driven by a small synchronous motor which is fed by a frequency-stabilized sine wave generator interrupts the two transmitted beams in such a way that only the main intensity of one beam a t a time is transmitted. The laser output power is adjusted to a level where both beams show the same output power (-700 mW); thus as a net effect, a laser beam with slightly varying intensity (at the frequency 2 Y , if Y is the chopping frequency for each beam), but with a switching wavelength impinges on the cell. The cell is a piezoelectric ceramic tube (PZT-5H, supplied by Vernitron; length: 76.2 mm, inner diameter 19.0 mm) which is sealed off at its faces: at one face with a quartz window, at the other with a ceramic plate incorporating a small quartz window and an opening to fill the cylinder. The piezoelectric ceramic acts simultaneously as a sample cell and as a pressure sensor for the pressure fluctuations induced in the sample liquid by absorbed radiation. This pressure sensor is advantageous for the detection of small pressure fluctuations in liquids as it is a live sound recorder and hence adjusted to liquids with their high acoustic stiffness. Alternatively, a crystal microphone might be used as well. Condenser microphones however, usually employed for opto-acoustic measurements in gaseous media are inadequate as they are sensitive primarily for the motion of its membrane. The amplitude of motion Ax is rather small in liquids for a given pressure fluctuation Ap according to Ax = Ap/2.nupc (where u = frequency, p = density, c = velocity of sound), as the density of ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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