bottle, and a statistical analysis was again performed on the resulting data (see Table I). The elemental concentrations and respective standard deviations thus obtained reflect the errors introduced by handling a large number of separate cell cultures. These larger standard deviations also reflect inherent variability of the cell cultures. Note that the concentration of Fe obtained, 101 ppm, is in agreement with the value calculated above using the intercept method.
CONCLUSIONS These results indicate that the application of PIXE to L-cell samples is most successful in determining concentrations of the elements K, Fe, and Zn. Less precise results are obtained for the elements Ca, Mn, and Cu. The modest precision observed for the 19 identically-grown cell cultures is reasonable, considering the inherent variability of biological systems. Work is now underway examining the variation of
L-cell trace-element content as a function of trace-element content in the nutrient medium.
LITERATURE CITED (1) T. B. Johansson, R. Akselsson, and S. A . E. Johansson, Nucl. Instrum. Methods, 84, 141 (1970). (2) K . Samsahl. D. Brune. and P. 0. Webster. I n t . J . A D D / .Radiat. Isot.. 16, 273 (1965). (3) P. A. KRos,R. Sinchir, and C. Waymouth, Exp. CellRes., 27, 307 (1962). C. J. Umbarger, R. C. Bearse, D. A. Close, and J. J. Malanify, Adv. X-Ray (4) Anal.. 16. 102 119731 - -, (5) M. Barretie, -G. Larnoureux, E. Lebel. R. Lecomte, P. Paradis, and S. Monaro, Nuci.Instrurn. Methods, 134, 189 (1976). (6) R. C. Bearse, D. A. Close, J. J. Mahnify, and C. J. Umbarger, Anal. Chern., 46, 499 (1974). .
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RECEIVED for review August 3, 1977. Accepted September 19, 1977. Work supported in part by the University of Kansas General Research Fund and by Biomedical Sciences Support Grant RR07037.
Pulse-Counting Techniques in Organic Mass Spectrometry Jedidja Freudenthai‘ and Laurens G. Gramberg National Institute of Public Health, Laboratory of Toxicology, P.O. Box 1, Bilthoven, The Netherlands
Puise-counting techniques in mass spectrometry are described with which it is possible to make accurate measurements (peak matches) In gas chromatographic peaks of only a few seconds wide. An accuracy of better than 1 p v in the mass determination can be obtained on a Varian Mat 731 mass spectrometer from an amount of less than 100 pg for mod substances. The accuracy at these levels is the result of the high sensitivity with which the mass spectrometric peaks are observed. Three different quantitatlve methods, using gas chromatographic inlets as well as direct inlet systems, are described. These quantitative methods are also based on pulse-counting techniques. The design for the electronic circuitry for a Varlan Mat 731 and a Varian Mat CH5 mass spectrometer is given.
In organic mass spectrometry, the determination of accurate masses of unknown compounds is often done with “peak match” procedures (1). There are restrictions in performing an accurate peak match. The unknown peak should be long enough on the oscilloscopic screen of the mass spectrometer, and the unknown compound should be present in sufficient quantity to create a peak with a reasonable intensity. When enough of the unknown compound is available, a peak match can be made using the direct inlet of the mass spectrometer. When the unknown compound enters the mass spectrometer via a gas chromatograph, the situation becomes questionable. If the gas chromatographic peak is wide, as,for example, from a packed column, a peak match might be still possible, provided enough of the compound is available. In the case of a narrow peak, as from a capillary column, the peak match is impossible. In this paper a pulse-counting technique will be described in which it is possible to peak match when the compound is present for only a short time. The integrating properties of
the pulse-counting system make it possible also to perform a peak match from much smaller quantities of the unknown substance. Both Properties make Possible an exact mass determination of unknown peaks in narrow gas chromatographic peaks.
PULSE-COUNTING TECHNIQUES In most applications in organic mass spectrometry, the signal is obtained via an electron multiplier. This signal consists of a series of pulses of only a few nanoseconds wide. The width of the pulses depends on the type of the electron multiplier used. In our experiment, we used electron multipliers supplied by Varian for the mass spectrometers Mat 731 and Mat CH5. The pulses are clouds of secondary electrons, created by the ions hitting the cathode of the electron multiplier. The ions from the mass spectrometer will arrive at the cathode of the electron multiplier randomly distributed in time, and consequently the pulses coming from the anode will do the same. There are several ways to handle this pulse-shaped signal. The normal way in organic mass spectrometry is to couple the anode of the electron multiplier to the high impedance input of an electrometer amplifier. The pulse character of the signal disappears, and the result is an averaged analog signal. Another approach is the pulse-counting technique. This technique is common in physics and physical chemistry, but not so in organic chemistry. In the pulse-counting technique, the single pulses (or ions) are counted (2-5). A current measurement consists of counting the number of ions in a certain time interval. This methodology has advantages over the electrometer methodology; it gives the maximum possible information about the signal with a minimum of noise. Since the data are already in a digital form, the data handling is easy to perform. A disadvantage of the pulse-counting system is that it cannot accept strong signals. With these signals, the interval between two pulses may become too small, such that the pulses are not separated anymore, Therefore a pulseANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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discriminator
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Tultichannel analyzer
in the multiscale mode. The timing is such that the periodic signal of the mass spectrometric peaks is stored on the same position in a memory subgroup of the multichannel analyzer
this condensor a saw tooth potential is applied, sweeping the ion beam in front of the exit slit of the mass spectrometer. The ratio of the accelerating voltages [and of electrostatic analyzer (ESA) voltages for double focusing instruments] of the mass spectrometer is then adjusted until the two peaks match, from which the accurate mass can be calculated. The procedure in the pulse-counting system is analogous to that of the usual peak-match procedure. The differences are that the signal is treated with pulse amplifiers and the data are represented on a multichannel analyzer. The storage of the unknown mass spectrometric peak is in one memory subgroup of the multichannel analyzer. The reference peak is stored in another memory subgroup. Display of the content of the two memory subgroups on the oscilloscopic screen of the multichannel analyzer makes a comparison between the two peaks possible (peak match). A somewhat similar methodology is described in the literature, where a time averaging computer (Varian TAC 1024) is used (6, 7). In this approach the signal from the mass spectrometer is in analog form and processed in the time averaging computer. The methodology with the time averaging computer is described for quantitative applications and not for peak matches on compounds which are present for a short time. The main advantages of a pulse-counting system over an analog system are the high speed and the better performance in signal to noise. For a peak match of a compound present for only a few seconds, these properties are important.
STORAGE OF MASS SPECTROMETRIC PEAKS I N THE MULTICHANNEL ANALYZER The storage of the periodical signal (scan) of a mass spectrometric peak in a memory subgroup requires a proper triggering of the multichannel analyzer. The commercially available multichannel analyzers do not possess the required triggering electronics. The timing of the triggering is given in Figure 2. The electronics designed for this purpose are given in Figure 3. The properties of the electronics should be such that a trigger pulse (“start”) w ill open the first channel of a memory subgroup a t the start of the sweep through the mass spectrometric peak. Another trigger pulse (“stop”) will stop the multichannel analyzer advancing through the channels in the memory subgroup in the multiscale mode. After this, the first channel of the memory subgroup will be addressed again with the next trigger start pulse. This periodic procedure repeats throughout the measuring time. The periodic signal of the mass spectral peak is stored and in-
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Figure 3. Electronics for triggering the multichannel analyzer. The input is a block pulse from a flip-flop in the decade bank electronics of the Varian Mat 731 mass spectrometer. The flip-flop switches when the slope of the sawtooth potential for the scope and the deflection condensor changes sign. The block pulse is then differentiated on the positive as well as the negative flanks. The differentiated pulses are shaped (in the first SN 74121) into a good trigger pulse to give the start trigger pulse. This pulse is delayed (in the 555). The variable delay time can be set with the potentiometer (100 K). This delayed pulse is shaped into a good trigger pulse (in the second SN 74121) to give the stop trigger pulse
tegrated in the memory subgroup. T h e same procedure can be used for the reference peak in another memory subgroup. Both reference and unknown peaks can be displayed on the oscilloscopic screen of the multichannel analyzer. If they do not match, the reference peak is erased and recorded again using a different accelerating voltage setting, this process being repeated until a match is obtained. In our experience the whole operation can be performed within 2 min. In contrast to the usual peakmatching procedure the stability of our mass spectrometer (Varian Mat 731) was such that it allowed the unknown and reference peaks to enter one after the other into two memory subgroups of the multichannel analyzer. Compared to the alternating procedure in the usual peak matching, this gives an additional gain of two in sensitivity. The stability of the mass spectrometer can be checked by entering a PFK peak in one of the four memory subgroups repeatedly over a 2-min time interval. A simultaneous display of the peaks should not show a shift in case of good stability. An investigation into possible sources of instabilities showed that the ESA voltages are usually highly stable. Instabilities may arise at the magnet. The reference resistor for the current regulator of the magnet should not be subjected to sudden temperature changes. Furthermore the magnet may be subjected to mechanical instabilities from pressure changes in the cooling water of the magnet. A closed circuit cooling system is a solution to this problem. Before a peak match is performed, the ratio of the ESA voltages is checked with the help of two PFK peaks with an accurately known mass ratio with a calibration facility on the decade bank of the mass spectrometer. It was found that the precision of the peak match with the pulse-counting procedure could be performed more precisely than with the conventional peak match procedure. This improvement revealed, however, that slight position changes of the peaks could be observed in time, apparently because the calibration of the mass spectrometer decade banks changed. Keeping the temperature changes in the decade banks as low as possible is the solution to this problem.
It must be noted that in the normal peak-matching procedure the same drift is present, but remains unnoticed.
RESULTS OF PEAK MATCHING As mentioned before, a peak match with the pulse-counting procedure can be performed with an accuracy better than with the conventional peak-match procedure. However, the main strength is that a peak match can be performed on a small amount of a compound which is present for only a few seconds, as in the gas chromatographic peak from a capillary column. With the Varian Mat 731 mass spectrometer, peak matches were made a t a resolution of 10000, 10% valley definition. For most substances, an accuracy of 1 ppm was obtained for an amount of 100 pg or less. An example of the peak shapes i s given in Figure 4 for hexachlorobenzene for the mass m l e = 284. At these small amounts of substance, the number of channels used in the experiments is only around ten. This number of channels was the best compromise between peak definition and peak height. In case of more substance, the number of channels in the peak can be increased in order to get a better peak definition. The shape of the peak is determined by the number of counts in the different channels. The variability in the numbers of counts can be obtained from ion statistics.
QUANTITATIVE MEASUREMENT WITH T H E PULSE-COUNTING SYSTEM With the pulse-counting system, the amount of substance coming from a direct inlet system or 11 gas chromatograph can be quantified. A mass spectrometric peak of the substance is stored and integrated in a memory subgroup in the same way as described before. The number of counts in the peak or the peak height can be used for quantitation purposes. The interesting feature of this type of quantitation is that the exact peak position is known also. Interfering neighboring masses can be detected. This quantitation procedure has been described in the literature for an analog input signal (6, 7). The advantage of the pulse-counting system compared to the analog method ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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Table I. Sensitivities and Standard Deviations of Mass Fragmentographic Measurements, Using Pulse-Counting Techniques Varian Varian Varian Mass spectrometer Mat 7 3 1 Mat 7 3 1 Mat CH5 Resolution, 10000 1000 1000 10%valley definition Emission currenta, mA 1.6 1.5 1.0 Electron energy, eV 100 1000 70 Amount sample (HCB), pg 1 2 0 2.5 2.5 Counts 700 10 400 2890 Countsipg HGB 32 280 4160 Re1 std dev 6 5 2.7 of peak area, % ’ The emission current for the Varian Mat 731 is the electron current on the ionization chamber. The emission current for the Varian Mat CH5 is trap current. All measurements were done on the m/e = 2 8 4 peak of hexachlorobenzene. The relative standard deviations of the gas chromatographic peak areas were obtained from ten measurements.
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