Anal. Chem. 1987, 59, 185-189
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Performance of a Hadamard Transform Photothermal Deflection Imager with Continuous Wave Laser Illumination Fotios K. Fotiou and Michael D. Morris* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
An expanded and line-focused Ar’ laser beam was used to irradiate samples on a TLC plate and create refractive index gradlents for transverse photothermal deflection measurements. The beam was spatially encoded with a Hadamard mask sequence. Ouantitatlve measurements of the absorbance of deposited sampies were obtained by inverse Hadamad transtormation of the raw photothermal signals. Results were comparable to single point measurements at similar delivered powers. Procedures for correction lor nonuniform pump laser power denslty and poSnloning or collimation errors in the probe laser were developed.
Transverse photothermal deflection (TPD) or mirage effect spectroscopy (1-5) is increasinglyemployed for ultrasensitive absorption measurements of solids and surfaces. The technique provides higher sensitivity than photoacoustic spectroscopy in an experimental configuration which is no more complicated. Transverse photothermal deflection is commonly performed with a laser source to take advantage of its high spectral brightness, monochromaticoutput, spatial coherence, or some combination of these properties. However, laser illumination is difficult to employ with many solid samples. Gross thermal decomposition is common, especially with high peak power pulsed lasers. To use a 351-nm XeF excimer laser as a transverse photothermal deflection source, we were forced to attenuate the 90-100 mJ pulses to about 2 mJ to avoid burning even robust samples (6). Less obviously, photochemical transformations, such as cis-trans isomerization, may be quite facile under laser illuminntion. This problem has already been observed (7). Since photochemical reaction may cause absorbance changes without obvious changes in color, systematic errors can go completely undetected. It is important to reconcile the need for high laser power or energy to maximize signal-to-noise ratio with the need for gentle irradiation to minimize unwanted sample transformations. “his problem has been recognized by several groups working in both photoacoustic and transverse photothermal deflection spectroscopies. They have all proposed techniques in which an unfocused or line-focused beam illuminates an extended region of sample. Fournier and co-workers employed a line focused argon laser to generate photothermal signals (8). After data collection with multiple orientations of the sample, the spatial distribution of sample was recovered with a tomographic back projection. Only preliminary data from that experiment are available. Coufal and co-workers demonstrated the feasibility of Hadamard spatial coding in photoacoustic detection (9). They employed 7 and 15 spatial element masks, prepared by photographing appropriate patterns onto glass plates. Using a glassy carbon sample, they demonstrated the feasibility of the technique. Recently, our own group introduced the use of Hadamard coding (10) for transverse photothermal densitometry employing a pulsed laser as light source. In those preliminary experiments a Hadamard mask was manually 0003-2700/87/0359-0185$01.50/0
shifted above the sample surface, using a micrometer head. Good results were obtained with samples of potassium chromate deposited on thin-layer chromatography plates. The photoacoustic and photothermal deflection experiments demonstrate the feasibility of distributed configurations. All previous studies have employed intensely absorbing samples, which are either homogeneous or consist of one or two absorbing regions on a nonabsorbing substrate. Important questions of sensitivity, linearity, and dynamic range have not been addressed. There have been no direct comparisons of the distributed signal technique to conventional point illumination photoacoustic or photothermal measurements. It is the aim of this study to characterize Hadamard transform photothermal deflection more completely. An argon ion laser was chosen as the pump beam. This laser provides a stable, well-regulated output intensity and excellent pointing stability. These properties make the argon ion laser more suited for technique characterization than a pulsed laser (11, 12). The major disadvantageof the argon ion laser is its nearly Gaussian beam cross-section. A spatially uniform cross-section would be preferred. We employ trans-azobenzene deposited on thin-layer chromatographic plates as samples. The same system has been used to characterize a conventional photothermal deflection densitometer (7,13). Hadamard transform infrared (HT-IR) spectroscopy was investigated in the later 1960s and early 1970s as a spatially multiplexed alternative to conventional IR spectroscopy (14). A spectrum was spatially dispersed in a conventional monochromator and the bands were observed simultaneously through a series of masks. A multiplex advantage was obtained. The technique was abandoned in favor of FT-IR spectroscopy (15, 16),which offered a larger multiplex advantage. Hadamard encoding remains an efficient method for spatially multiplexing certain kinds of signals. For example, Hadamard encoding has recently been employed in a spectrometer based on an array of light-emitting diodes and designed for measurements of trace atmospheric species (17). Our experiments are different in principle from the HT-IR technique. Our goal is to spatially distribute the laser light across the imaging area. For this application, Hadamard masking is an efficient method to encode the source light. The old Fourier vs. Hadamard arguments do not apply. There is no conventional multiplex advantage to be obtained in a photothermal deflection experiment. There is a “spatial distribution* advantage, only. Hadamard transform photothermal deflection requires that photothermal signals be additive along a line and independent of the position along that line. These assumptions have been incorporated into photothermal deflection theory (I, 2). The assumptions were implicitly accepted for tomographic measurements of Fournier and co-workers (8) and for our own preliminary Hadamard transform measurements. We have explicitly tested and verified the assumptions on a simple two spot system (7).
THEORY If the excitation beam is line focused coincident with a probe beam parallel to a flat surface, the resulting PD signal will 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 1. JANUARY 1987
be the sum of signals from each resolution element along the line. Thus,when a mask consisting of opaque and transparent slits intercepts the pump beam,the measured signal generated by mask j is given by
Here y j is the signal from the j t h mask, sj = (slj, s ~ j..., , snj) is the vector of mask elements of the j t h mask and xi is the signal at the position i on the sample. The element sjj has the value 1if he corresponding ith resolution element is illuminated and 0 if it is not illuminated. For n resolution elements, the measurement process requires a sequence of at least n different masks. If the n masks are properly chosen, they define a system of n linear independent equations which completely describe the system
90
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In matrix notation, the set of eq 2 may be written as Y = s.x (3) The system can be solved by calculating the inverse of the matrix S, according to x = s-1.y (4) The properties of S matrices and techniques for their generation have been discussed in detail (14). An S matrix can he cyclic. A cyclic matrix has the property that each row is generated from the previous one by shifting its elements one position to the left (or right) and placing the overflow in the position of the element that was first shifted. The advantage of using a cyclic matrix is that a single mask of 2n - 1 slits can be used to generate the configurations of all n individual masks. The mask is shifted incrementally underneath a limiting aperture (or frame) a distance of one slit width. Each shift of the mask generates another row of the S matrix. Shifting the mask n - 1times generates the entire S matrix sequentially. Recovery of the image is carried out by multiplying the vector of the measured signals by the inverse of the S matrix. The inverse SF is computationally simple to generate and is given hy s-I
= -.w n+l
(5)
Here W is a matrix which has -1's where SThas 0's and +l's where SThas +1'a Furthermore for one class of matrices constructed from the -maximal length shifbregister" sequence (14) the multiplication procedure can be done according to the fast Hadamard transform (FHT) algorithm which reduces the number of additions and subtractions required from n(n - 1) to n log, n 2n. The FHT can be used only if n = 2k - 1, where k is an integer.
+
EXPERIMENTAL SECTION The samples were known quantities of trans-azobenzene, deposited from hexane solution ou Merck silica gel 60 HFTLC plates, as previously described (7).For most experiments the azobenzene spots were not subjected to chromatographic development. For comparison to a conventional TPD system, the previously described (7)chromatographic procedure was used. The optical rearrangement for these experiments is similar to that used previously (IO)and is illustrated schematieallyin Figure
1. A Lexel85-1 Ar+ laser was used as the excitation source. In most experiments, the laser was operated at 488 nm with power of 15 mW delivered to the sample surface as a line 14 mm long. Measurements of developed plates were carried out with 4.5 mW of delivered power at 458 nm. A 1-mW He-Ne laser was used to probe the refractive index gradient above the TLC plate. The distance from the center of the probed region to the knife edge was about 50 cm. The argon ion beam was shaped by two cylindrical lenses. A 4-cm fd length lens (L,) expanded the beam along the direction of the He-Ne laser beam. A 30-cm focal length lens (L,)condensed the beam to an approximate line parallel to the He-Ne beam. The two lenses were 26 em apart, with L, 32 em from the sample. A mechanical chopper was used to modulate the pump beam, usually at 7 Hz. The deflection of the probe beam was measured with a knife edge and photodiode. The PD signalswere amplified with an Ithaco 1201 preamplifier and demodulated with an EG&G/PARC 5101 lock-in amplifier. The lock-in was operated with a 0.1-s time constant and 12 dB/octave attenuation. Data from the lock-in amplifier were digitized to 12-bit resolution and stored on a small computer, as described below. The 69-element cyclic Hadamark mask previously described (IO)was used. The mask provided 35 0.406 mm spatial resolution elements. The mask was mounted on a translation stage and positioned below a fixed limiting aperture. The translation stage was moved with an Oriel 18269 micrometer head equipped with a dc motor and shaft encoder. To limit the data rate and avoid computer errors, the encoder pulses were scaled 1:lO with a SN7490 counter. The sealed pulses represented 1-pmincrements, at a maximum frequency of about 210 Hz. A PDP 11/03 computer was used for data acquisition, motor control, and inverse Hadamard transformation of the data. All programs were written in FORTRAN. To position the stage, power to the motor was stopped in advance of the desired location, and the motor was allowed to coast to a stop. Any resulting positioning error, always less than 9 pm, was used to correct the travel distance of the stage in the next positioning. Data were taken when the motor had come to a complete stop. In some cases, five or six data point, at 0.1-s intervals, were averaged. In another mode, the motor was moved continuously. In this case, one measurement was taken each time the mask had been moved by one position, 406 pm, or 406 encoder pulses. A simple normalization procedure was developedto correct for pump laser beam intensity distribution and probe laser beam divergence or misalignment. Six lWng spots of trans-awbenzene were deposited on a plate at 2.5-mm intervals. The signals from this system were measured and transformed. The plate was then shifted 0.406 mm along the probe direction and the measurements were repeated. The area under each peak was measured, and a Gaussian regression was used to yield the calibration curve. The coefficients of this equation were used to correct experimental data.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
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RESULTS AND DISCUSSION The raw data from a plate spotted with 100-ng samples of trans-azobenzene, deposited a t 2.5-mm intervals along a straight line, is shown as Figure 2. Because of the uniformity in spot spacing and composition, the data show some periodicity. In general, plots of raw data appear as almost randomly fluctuating signals. In most cases the plots are not visually related to the sample image. The transformed data from the sample of Figure 2 are plotted in Figure 3A. The inverse Hadamard transform required about 0.56 s of CPU time on our computer. The spot separations in the transformed data are equidistant. The peak maxima are 6 mask width units, 2.44 mm, apart. The data are in perfect agreement, within experimental error, with the actual spot separations, 2.5 f 0.5 mm. However, the relative intensities are not proportional to the amounts taken. The data clearly show that the photothermal response is not uniform across the plate. In our experiments the sample region probed is about 1.4 cm long and the sample to knife-edge distance is 50 cm. Because we are taking the absolute deflection of the laser beam as a measure of the angular deflection, there is a systematic variation in sensitivity along the probe region. The signal from a uniform sample should decrease 2.8% from the end of the sample farthest from the knife-edge, at 1.5 mm in Figure 3A, to the end closest to it. This small systematic change cannot account for observed nonuniform response. There are two other possible sources of nonuniform response. First, the pump beam intensity is approximately Gaussian, not uniform, along the line of the measurements. Second, the probe beam may not be perfectly collimated or perfectly parallel to the plane of the plate. Nonuniform pump beam intensity is an obvious source of systematic error. Probe beam collimation and alignment have previously been shown to be important in maximizing signal/noise ratios in transverse photothermal deflection measurements (1, 7). We attempted a correction for the pump bean intensity profile. The intensity profile of the line-focused beam was measured independently and the local intensity values were used to normdize the measured signals. This procedure improved the relative intensity values, but the standard deviation in a set of five measurements was flO.5%. Better results were obtained with an alternative procedure. A single spot of trans-azobenzene was deposited on a TLC plate. The plate was scanned in the conventional manner (7)with the Hadamard mask removed. The variation of the PD signal with distance was used to correct the transformed signals. This procedure reduces the standard deviation to about A3.8%. However, a second motor-driven stage is needed.
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Ultimately, the simple calibration procedure described in the Experimental Section was found to be satisfactory. With six 100-ng spots of trans-azobenzene, as shown in Figure 3A, 12 data points are obtained. The fitted Gaussian curve is shown as Figure 3B. Figure 4A shows the transformed but uncorrected data from a plate spotted with 75-, loo-, 125-, and 150-ng spots of trans-azobenzene. Application of the corrections from this procedure to the data of Figure 4A yields the corrected peaks shown as Figure 4B. The slope standard deviation for a linear least-squares fit of the areas of these eaks against amount taken was f3.8%. Examination of Figures 3 and 4 also shows that the transformed signals are noisier at the edges of the image area than in the center. This behavior was always observed and results from the nonuniform power density in the argon ion laser. The power density at the center of the distribution was a factor of 3 larger than the power at the edges. The linear dynamic range of the system was studied. The transformed PD signal was found to be linear with amount taken from 5 to 280 ng (correlation coefficient, 0.996;standard error of slope, *3.5%). Below 5 ng, reliable quantification was impossible. Above 300 ng, the calibration curve became concave downward, probably reflecting the nonlinearity which occurs with increasing sample absorbance. Similar behavior is observed with a scanning photothermal deflection system (18). The working curve had not reached a constant value a t the largest sample taken, 500 ng. The modulation frequency response of the PD of 0.5-wg spots was studied over the range 7-145 Hz. The signal is approximately inversely proportional (slope -0.9, log-log plot) to frequency (correlation coefficient, 0.993; standard error of slope, f4.2%). In our previous study (7)the signal decreased more slowly (slope -0.6) a t low modulation frequencies.
188
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987 IM
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Figure 4. (A) Transformed data from four spots 75, 100, 125, and 150 ng of trans -azobenzene. (B) Normalized data.
However, the present study was conducted at significantly lower power densities, and we cannot rule out the presence of nonlinear behavior in our earlier work. The power dependence of the signal intensity was measured. The signal increased linearly with the pump beam intensity (correlation coefficient, 0.997;standard error of slope, rt3.0%), over the range tested, 0.23-15 mW, as expected from theory ( 1 , 2).
The sensitivity of the system was compared to the sensitivity of the single element system described earlier (7). With 4.5 mW at 458 nm delivered to the plate and six data points per sample element averaged, the detection limit ( S I N = 2) for a sample of azobenzene in the center of the laser beam was 16 ng, measured against the noise in the base line. In the single element system, we obtained a detection limit of 1.3 ng with 21 mW delivered to the sample and base line noise averaged over 300 s. At the low laser powers employed, the dominant noise source is the fixed probe laser noise (7). The detection limit should depend inversely on the power delivered and on the inverse of the square root of measurement time. The linearly projected detection limit at 21 mW and 300 s is about 0.9 ng. Therefore, the Hadamard transform system compares quite favorably to the single element system in detection limit. We investigated data acquisition with continuous motor movement. In these experiments data were simply acquired each time the continuously moving mask reached the correct position, every 0.406mm. Figure 5 compares the results from the incremental movement and the continuous movement configurations. Figure 5A shows nonormalized data from three 50-ng trans-azobenzene spots, with incremental mask movement. Figure 5B shows data acquired from the same sample, obtained with continuous mask movement. In the continuous movement experiment, a point was acquired within 1ms of sensing the correct shaft encoder count. Since the maximum encoder pulse rate was less than 210 pulses/s, data were acquired within 1 pm of the correct mask
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position. However, because the lock-in amplifier time constant was 0.1 s, some systematic lag was always present in the data. The mask is shifted across one mask width in about 2 s, approximately 20 time constants. Therefore, the lag is not serious. We have not identified all of the noise sources in the Hadamard transform experiment. The mask positioning and drive system employed could be greatly refined. Their contribution to system noise may be substantial. The pump laser power is low enough (21 mW) that probe laser noise is still important. The measurements were made with a 0.1-s time constant. Noise from probe intensity fluctuations and pointing errors are important in the low hertz region. Like a Fourier transform, a Hadamark transform redistributes noise (14). In particular, noise spikes, which might arise from air currents or sudden changes in probe power supply output, are distributed throughout the spectrum. Detailed studies of these factors have not yet been carried out.
CONCLUSIONS Our studies demonstrate that Hadamard transform transverse photothermal deflection provides data that are similar in quality to that obtained in a single-element photothermal deflection experiment. The technique provides a practical solution to the problem of photochemical or thermal sample damage. Even though the fast Hadamard transform was not used in these experiments, computation time was only 0.5 s. Similar computation times would be required with most common laboratory microcomputers. Use of the fast Hadamard transform would be required for masks containing more than about 100 elements. At any practical mask length, computation time is unlikely to be more than a few seconds. No extraordinary effort is required to align the system. The masks do not have to be very precisely positioned under the aperture. An initial mask misalignment merely defines a new
Anal. Chem. 1987, 59, 189-193
set of spatial elements. Misalignment simulationson collected data verify that misalignment by as much as two elements effects primarily samples at the edges of the imaging area. The same conclusion has been reached by Plankey et al. (19). It is important, however, to preserve a mask alignment from run to run, in order to use one set of calibration data. Continuous mask motion is quite practical for use with CW laser sources. Incremental motion may prove more suitable with pulsed laser sources. Pulsed lasers typically have worse beam pointing stability than the argon ion laser used here. Some data averaging at each mask position might be desirable to correct for this problem. Few laser beams have a flat intensity profile. Correction for nonuniform intensity has been shown to be feasible. Alternatively, the laser beam can be shaped to have an approximately uniform intensity (20,21). A constant intensity profile beam would generate position-independent detection limits. Beam shaping might be the most practical alternative for use with dye lasers. The intensity profile usually depends upon the dye employed and ita concentration and a calibration curve for each dye employed would otherwise be needed. Registry No. trans-Azobenzene, 17082-12-1.
LITERATURE CITED (1) Jackson, W. 6.; Amer, N. M.; Boccara, A. C.; Fournier, D. Appl. Opt. 1981, 2 0 , 1333-1343.
189
(2) Murphy, J. C.; Aamodt, L. C. J. Appl. Phys. 1980, 57, 4580-4588. (3) Skumanlch, A.; Fournier, D.; Boccara, A. C.; Amer, N. A. J. Appl. P h y ~ 1986, . 59, 787-795. (4) Varlashkin, P. 0.; Low, M. J. D. Appl. Speclrosc. 1986, 40, 393-397. (5) Morris, M. D.; Peck, K. Anal. Chem. 1986, 58, 81lA-822A. (6) Fotiou, F. K.; Morris, M. D. Appl. Specfrosc. 1988, 40, 700-704. (7) Peck, K.; Fotiou, F. K.; Morris, M. D. Anal. Chem. 1985, 5 7 , 1359-1362. (8) Fournier, D.; Lepoutre, F.; Boccara, A. C. J. Phys. Colloq. 1983, C6, 479-482. (9) Coufal, H.; Moiler, U. Appl. Opt. 1982, 2 1 , 116-120. (10) Fotiou, F. K.; Morris, M. D. Appl. Spectrosc. 1986, 40, 704-708. (11) Fang, H. L.; Swofford, R. L. Ulhasensifive Laser Spectroscopy; Kiinger, D. S., Ed.; Academic: New York, 1983;pp 175-182. (12) Long, G. R.; Blaikowski, S. E. Anal. Chem. 1988. 5 8 , 80-86. (13) Masujima, T.; Sharda, A. N.; Lloyd, L. 6.; Harris, J. M.; Eyring. E. M. Anal. Chem. 1984, 56. 2975-2977. (14) Harwit, M.; Sioane, N. J. A. hdamard Transform Optics; Academic: New York, 1980. (15) Vanasse, G. A. Appl. Opt. 1982, 27, 189-195. (16) Hirschfeld, T.; Wyntjes, G. Appl. Opt. 1973, 72, 2876-2880. (17) Sugimoto, N. Appl. Opt. 1986, 2 5 , 863-885. (18) Chen, T. I.; Morris, M. D. Anal. Chem. 1983, 5 6 , 19-21. (19) Plankey, F. W.; Glenn, T. H.; Hart, L. P.; Winefordner, J. D. Anal. Chem. 1974, 46, 1000-1005. (20) Veidkamp, W. B. Rev. Sci. Instrum. 1982, 53, 294-297. (21) Grojean, R. E.; Feidman, D.; Roach, J. F. Rev. Sci. Instrum. 1980. 5 1 , 375-376.
RECEIVED for review July 10,1986. Accepted September 16, 1986. This work was supported in part by Grant GM37006 from the Public Health Service and in part by Grant CHE8317861 from the National Science Foundation.
Computer-Based Mass Measurement of Fragment Ion Spectra from Tandem Magnetic Sector Mass Spectrometers with an Electrically Floated Collision Cell Robert K. Boyd,*' Peter A. Bott: Brian R. Beer: Don J. H a r ~ a nand , ~ J. Ronald Hass3 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Methodology Is described whereby fragment Ion spectra, obtained from tandem magnetlc sector mass spectrometers, may be acqulred, mass-measured, and processed by using standard computer-based data systems. The mass spectrometers may be single or double focusing, and the cditsion cell In whlch the precursor Ions are colllsknalty activated may be floated from ground potential. The technlque has been evaluated wlth a tandem doublefocusing mass spectrometer to investlgate fragmentations of Ions derived from perfiuorokerosene (electron ionlration) and from sodium Iodide (fast atom bombardment lonlzatlon). Such fragment Ions have unambiguous atomic compositions, so that accuracy and preclslon of the assigned masses may be Investigated. Examples of application of the method to peptides are presented.
Tandem mass spectrometer has recently come of age as an established technique of analytical chemistry ( I ) . While Present address: Atlantic Research Laboratory, National Research Council of Canada, Halifax, Nova Scotia B 3 H 321, Canada. Present address: VG Analytical, Ltd., Wythenshawe, Manchesteri England M 2 3 9LE. Present address: Triangle Laboratories, Research Triangle Park, N C 27709. 0003-2700/87/0359-0189$01.50/0
Fourier transform ion cyclotron resonance techniques have considerablepotential in all aspects of masa spectrometry (2), the high-performance mass analyzer of choice is still the double-focusingmagnetic sector/electric sector combination. This is particularly true for applications to large molecules of biological interest. A tandem mass spectrometer has been described (3) in which both precursor ion selection and fragment ion analysis are achieved by using double-focusing analyzers. The success thus achieved (3) has led to the commercial availability of such instruments. It was recognized from the outset (3)that computer-based acquisition of the fragment ion spectra thus obtained would be advantageous. However, until recently it was not possible to apply conventional time-to-mass conversion algorithms, requiring internal calibrant masses, to fragment ion spectra obtained from those tandem instruments that use magnetic sectors for analysis o f both precursor and fragment ions. Essentially, this is because the ensemble of ions, formed in the ion source from a standard calibrant sample, cannot be transmitted in its entirety to the second (fragment ion) analyzer, but is first filtered by the magnet used to select the precursor ion. (This limitation clearly applies regardless of whether or not one or both magnets form part; of a doublefocusing combination.) A solution to this problem has been proposed ( 4 ) , whereby mass calibration of the scan function of the second magnet is achieved by use of a standard pre0 1986 American Chemical Society
