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Anal. Chem. 1W4, 56, 1474-1481
Minicomputer-Based Multichannel Signal Averager for Acquisition of Weak and Transient Mass Spectra Charles R. Snelling, Jr.,l J. Carter Cook, Richard M. Milberg, Mark E. Hemling, a n d Kenneth L. Rinehart, Jr.* School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801
A versatile hardware/soflware multichannel slgnai averaging system employing ion counting techniques has been developed for the acquisition of weak and transient mass spectra. The system has been successfully used to acquire hlgh- and low-resolution spectra from field desorption (FD), fast atom bombardment (FAB), electron ionlration (EI), and chemical ionization (CI)runs with accuracies averaging 5 ppm for high-resolution measurements. I n addition, the system has proved useful in acquiring high-resolution and isotope ratio data from gas chromatography/mass spectrometry (GC/MS) runs.
Mass spectrometry is one of the most sensitive analytical techniques available to the chemist, with significant structural information frequently being obtained from less than a microgram of material. However, in extending the technique to lower detection limits and higher masses, the mass spectrometrist is often faced with the problem of measuring accurately extremely small or fluctuating ion currents from limited samples. The problem of ion statistics is particularly acute in carrying out high-resolution field desorption and fast atom bombardment mass spectrometry (HRFDMS, HRFABMS) a t high masses. It was recognized early (1) that an averaging system would be of some use, and in 1970 a computer was employed to acquire and average multiple scans (2). In 1978 Brent (3) described a computer-averaged transient (CAT) system to facilitate high-resolution FD peak matching. Similar CAT systems have been used by Hass (4) and Van Ness (5)to lower the detection limits of dioxins and chlorinated pesticides, respectively. Multichannel averaging has also been used by Schulten (6) for quantifying FD by stable isotope dilution. Most recently, Ligon (7,8) has reported the application of a hardware-based multichannel analyzer for recording transient low-resolution FD spectra at high mass as well as high-resolution FD data. All of these reports describe hardware systems dedicated to specific purposes. We have recently developed a versatile hardware/software multichannel signal averager (MSA) for the real-time acquisition and display of mass spectral scans (9). The hardware portion employs a DEC PDP-8A minicomputer and an ion counting interface. The software is independent of the type of mass spectrometer employed and provides for the averaging of multiple scans, manual or automatic centroid calculations, determination of peak areas, Savitzky-Golay peak smoothing (1O);and accurate mass determination. In the present paper, we describe the required hardware and software and provide examples of the application of this system to high- and lowresolution mass spectrometry employing EI, CI, FD, and FAB imization modes, as well as to GC/MS and isotope ratio analysis. EXPERIMENTAL SECTION Overview of System. The MSA was designed as a software-based system so that parameters could be changed readily, Present address: Exxon Research and Development Laboratories, P. 0. Box 2226, Baton Rouge, LA 70821. 0003-2700/84/0356-1474$01.50/0
as dictated by the particular experiment being performed. This allows the operator to optimize such variables as the number of channels and the length of scan. A software-based system is also more transferable, allowing for quick translations as instrumentation in the laboratory is upgraded. The software can easily be modified to accommodate new experiments or additional postexperiment data manipulation. An overall diagram of the system is shown in Figure 1. The computer memory is divided into two equal segments, one for the acquisition of sample data and one for reference data. Each segment is further divided into channels representing time intervals in the mass scan. As each scan is taken, the new results are summed with the contents of the memory so that the spectra in memory are an average of spectra acquired. The MSA employs accelerating voltage scanning on instruments not equipped with field regulated magnets, but it can employ magnetic scanning with field regulated magnets. The narrow scan circuitry on the mass spectrometer’s display unit is used to generate a highly reproducible scan, generally of 5 to 10 s duration. In this work, using the accelerating voltage scan, the maximum mass range that can be scanned is 10% below to 10% above the nominal mass setting of the magnet and is limited only by the mass spectrometer’s particular scan circuitry. Mass Spectrometers. The MSA has been used with Finnigan MAT 731 and 311A spectrometers as well as with a VG 7070H and should be usable with any double-focusing instrument which allows reproducible accelerating voltage scans. Interface Hardware. The interface consists of an amplifier/discriminator and a counter/adder module. The amplifier/ discriminator (Model AD6, Pacific Precision Instruments, Concord, CA) is connected to the secondary electron multiplier (SEM) by a short cable. The discriminator level is set at 30 mV (determined empirically) to reject shot noise and the unit has been modified to produce TTL pulses of 60-11s width. The amplifier/discriminator converts the output of the SEM to TTL pulses which are counted by a 24-bit counter. The counter/adder logic is diagramed in Figure 2. The 24-bit adder is used to sum data for each channel as it is latched from the counter, resulting in a dead time of only 400 ns. The clock controls dwell time and the address decoder is used to control transfer of data to and from the PDP-8A computer via a 12-bit data bus. Each word that is transmitted contains a four-bit mask (including one bit left available for future development) to identify it to the interface with eight bits for information. Three data words are employed to provide a range of P4. Computers and Software. The MSA’s software consists of two main programs. The first, ASMS, written in OS-8 assembly language and residing on a DEC PDP-SA, controls the acquisition and real-time display of data from the mass spectrometer. Because of the difficulties associated with doing floating point calculations in OS-8 assembly language, a second program, NEWMSA, has been written in FORTRAN and stored in a DEC VAX 11/780. This program is used for the actual workup and calculations of MSA data. A third, minor program, PDPTOVAX allows for the transfer of data from memory on the PDP-8 to disk storage on the VAX. Both ASMS and NEWMSA are menu-driven with built-in descriptions of all commands and useful diagnostics. Documented software is available from the authors. The initiation of an acquisition requires the operator to specify the scan time and the number of channels, which will be used to determine the dwell time. Two clock words are transferred to the interface to control dwell time and the acquisition begins with a start-of-scansignal from the mass spectrometer. Data are plotted in real time on a VT-55 terminal and the dwell time calculation allows 0.3 s for autoranging calculations at the end 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
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I L
After the acquisition has been completed, centroids can be interactively determined with the data on the PDP-8A, using horizontal and vertical expansion as necessary, or the data may be transferred to the VAX where smoothing with a nine-point Savitzky-Golay (IO) routine and automatic calculation of masses and centroids may be carried out. Procedure. In order to use the exact mass equation (see Theory and Equations below), it is necessary to bracket the unknown peaks with two reference peaks. Once a suitable reference compound or mixture is chosen, the mass spectrometer is set up for an accelerating voltage scan which will encompass the three peaks, and the computer and interface are signaled by introducing the scan time and number of channels desired. Since the sample spectrum is often transient or weak, it is generally acquired fist. Scans are accumulated until the desired SIN ratio, which can be monitored on the VT-55 terminal in real time, is obtained. If, in an FD experiment, for example, the sample is depleted before a suitable SIN is obtained, the emitter can be reloaded and additional scans accumulated. The reference spectrum is then acquired by using any available ionization method, typically EI. In favorable cases, this dual acquisition process may take as little as 5 min. After both the sample and reference spectra are acquired, the data are handled as described under Computers and Software above. Single Reference Peak Method. Generally the mass range scanned is on the order of 1to 10 amu, but it can be as large as 20% of the normal magnet mass setting. For Inw-resolution measurements, the larger mass ranges pose no lems. The mass range used in a high-resolution measurem however, is determined solely by the particular reference stai .rd used and the difference in mass between its peak and that :the sample. By use of standards such as Fomblin or phosphazenes ( 1 1 ) with few fragment ions, this mass range may be as large as 50 to 100 amu. Scanning the accelerating voltage over such large ranges can result in source tuning differences across the mass range, causing mass discrimination as well as changes in peak shape and, hence, resolution. In such cases we employ a method in which a single reference peak is used, thus greatly reducing the mass range. A dual channel peak matching unit with an accuracy of better than 1ppm is used to produce the two reference peaks. With the first channel (A),
T A R
ELFCTRON MULTIPLIER
____
T
I
- -_-J
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+
24-BIT DATA LATCH
24-BIT A D D E R
CLOCK
t t 1/0 BCFFER
VAX
3 2 K !(EMORY
t
I
cu7 DISPLAY
HARDCOPY
Flgure 1. Overall diagram of MSA system.
of each scan. The acquisition may be stopped after any scan and later resumed, which allows for reloading emitters, retuning, etc.
’8’
’
: CLK
c
RISTATE
B CLK TRISTATE.
ADDRESS 3 DECODER ADDRESS BITS
CLI CL2 DATO I DAT02 DATO3 INDAI INDA2 IN DA3
Flgure 2. Diagram of the logic Units used in the interface.
1475
,
7 OUTE INDAIEN
‘8
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‘8 INDA2-
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’
: TRISTA
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
w
50
0
Flgure 3. Schematic representation of the single reference peak method: (a) original reference peak (MJ appearing at accelerating voltage V,, sample peak at M, in both a and b; (b) origlnal reference peak shifted to higher mass (M,) by changing accelerating voltage to v2.
0
0,5
I .o
1.5
[(M~-M,)/M J
2.0 io0
2.5
3.0
Flgure 5. Comparison of the mass calculated using eq 1 and 4 as a function of the separation between the two reference peaks.
sumed. However, since the mass of an ion is inversely proportional
B2R2 m/z = 2v
to the accelerating voltage (eq 2) and a linear scan results in an exponential mass function, a correction must be made which involves the accelerating voltage a t each of the masses during the scan. Ligon’s expression (12) for the real mass of the unknown, Mx, in terms of only three variables, ML, MH, and MI, can be simplified to TL
Tx
(3)
TH
Flgure 4. Schematic drawing for the determination of an unknown mass using linear interpolation.
a reference peak of lower mass is chosen to be as close to the unknown as possible (Figure 3a). With the second channel (B), the accelerating voltage is decreased to a point where the original reference peak is at higher apparent mass than the unknown (Figure 3b). The two reference peaks are then acquired, with the peak matching unit automatically switching between the A and B channels, so that the reference spectrum in the computer will contain both peaks. The mass of the new (artificial) reference peak can be easily calculated from the mass of the original reference peak and the ratio of the two accelerating voltages. In many cases, the second reference peak can be generated above the sample peak by decreasing the accelerating voltage by as little as a few hundred parts per million. At mass 500, this means the mass range to be scanned can be reduced to 0.2 to 0.5 amu. This decrease in the mass range represents a 2- to 5-fold decrease in the optimum number of channels (ONC, cf. below) as compared to the two-reference-peak method and allows faster scans or increased sensitivity (longer dwell time) for a given scan speed.
THEORY AND EQUATIONS Exact Mass. To determine the nominal or accurate mass of an unknown, the MSA uses an interpolation scheme in which the unknown is flanked on both the high and low mass sides with reference peaks of known mass (Figure 4). The mass of an unknown can be expressed by
MI = ML
+ TTHx -- TTLL
( M H- M L )
(1)
where ML, M I , and MH are the masses of the low-mass reference, unknown (by interpolation), and high-mass reference, respectively, with corresponding centroid times T L , T,, and TH, and a linear relationship between time and mass is as-
Combining eq 1 and 3 gives
for the mass of the unknown peak, corrected for the accelerating voltage scan’s nonlinear mass scale, in terms of the known or directly measurable quantities, mass and time. The mass calculated by simple interpolation using eq 1 differs from that calculated by using eq 4 as shown in Figure 5, where the mass measurement accuracy is plotted against the magnitude of the mass range scanned. For a mass range larger than 1%,the error associated with the simple interpolation scheme becomes unacceptably large (>25 ppm), while for small mass ranges (< 0.5%) the mass accuracy using simple interpolation is better than 6 ppm. Optimum Number of Channels (ONC). The accuracy of any mass measurement depends on the number of data points collected across the peak. The larger the number of points collected, the closer they approximate the true peak shape, leading one to use as many channels as possible for every acquisition. However, two other factors need to be considered. The fixed amount of memory available for data storage means that for a given number of channels, as the number of channels per peak is increased, the mass range must be decreased. Second, for a given scan time, the dwell time must decrease as the number of channels increases. The proportional increase in the number of scans and, therefore, in sample lifetime required to maintain constant sensitivity is antithetic to our reasons for developing this MSA system. Calculation of the ONC according to ONC = (number of channels per peak X mass range X resolution)/average mass (5)
ANALYTICAL CHEMISTRY, VOL. 56. NO. 8. JULY 1984
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ZcrvomicinnA ( M +No1 Singie Oscillogrnphic Scnri Resolullon - 1300 Electron Multirl~er=l x108
M
C
Figure 6 . Effect of successive Summation on peak shape and definiton 01 the m / z 59 ion of acetone in the F I mode: (a) single Scan from oscilloscope; (b) single MSA scan 01 Same peak: (c) four MSA scans; (d) ten MSA scans. allows one to specify the number of channels that will be used to determine the peak shape. The mass range is calculated in parts per million and then divided by the average peak width to give the number of peak widths in the mass range. This is then multiplied by the user-defined number of channels per peak to afford the ONC. In the present work, 5 to 10 channels per peak were used for low-resolution determinations when large mass ranges were scanned, while 20 to 30 channels per peak were used for high-resolution measurements. RESULTS AND DISCUSSION The dramatic increase in SIN and definition of a typical weak FI signal achieved by employing the MSA can be seen in the series of photographs in Figure 6. The fimt photograph (Figure 6a) shows the very weak and poorly defined signal of the m / z 59 ion of acetone taken directly from the oscilloscope display. By comparison, the second photograph (Figure 6b) is a single scan of the same peak using the MSA. Note that the peak is still poorly defined due to ion statistics. The third photo (Figure 6c) represents a summation of four MSA scans. At this point, the peak has been defined sufficiently to see that it is actually a doublet. After ten MSA scans (Figure 6d) and one autoranging, a doublet is clearly discernible with a symmetrically shaped major peak and a much improved S/N ratio. The total time required to obtain these ten MSA scans was 60 s. Similar results, using the MSA to acquire low intensity and short-lived spectra, are presented below. Low Resolution. It has been very difficult in the past to obtain good low-resolution or high-resolution FD spectra for several classes of compounds, including peptides of high molecular weight and/or polarity. A good example of these peptides of high molecular weight is provided by the zervamicins, members of the peptaihophol class of antibiotics which are thought to effect ion transport through cell membranes by pore formation (13-15). However, they are not amenable to ordinary peptide sequencing techniques such as Edman degradation and enzymatic cleavage because of their
-
NO 1847
13"
cnonne, f
230
300
Figue 7. Lowresolution FD spearurn of zewamicin I I A (1): (a)single oscillographic scan; (b) sum of five scans using MSA.
blocked N termini and C termini and their high concentration of a-methylamino acids such as a-aminoisobutyric acid (Aib). Mass spectrometry has, therefore, become the method of choice in determining the amino acid sequences of this type of peptide (16). The zervamicins were particularly intractable by FDMS and only recently have their structures been determined through the use of FABMS (17). The top spectrum, Figure l a , represents a single oscillographic trace, the best low-resolution FD spectrum obtained to date for zervamicin IIA (1, Figure 7). Even after the sample was doped with sodium chloride to enhance the molecular ion (28), a gain of 10s was required on the electron multiplier. The effects of sample sputtering from the FD emitter and ion statistics account for the poor peak shapes and SIN ratio. The bottom spectrum, Figure 7h, represents the sum of five scans obtained by using the MSA. Both the quasi-molecular ion at m / z 1847 (M + Na) and the (M + Na - H,O) ion at m / z 1829 are seen clearly. The peak shapes are much better since five times the number of ions are represented (statistid and the sample sputtering effects have been averaged. The two reference peaks obtained in the E1 mode to calibrate the FD spectrum were m/z 1829 and m / z 1845 of Ultramark 2500. The total time needed to acquire the five MSA scans was less than that needed to obtain the single oscillographic scan in Figure la. Many substances, including organometallic compounds, exhibit facile fragmentation from the molecular ion in the FD mode, a situation which comes ahout when the energy needed to break a particular bond is similar to the energy needed to desorb the sample. Neutral losses from the molecular ion may include halogen, acetic acid, and carbon monoxide. Generally, molecular ions for these compounds are not seen. The FD spectrum of a chlorinated copper iridium complex, 2 (C,,H,&ICuIrO6P2). is shown in Figure 8. The spectrum was obtained by using the MSA to scan repetitively the mass range m / z 1030 to m / z 1115. While approximately 15 scans were being obtained, the emitter current was increased from 0 to 23 mA by approximately 5-mA increments and held at
,
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
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Low Resolution FD vs El Resolution = 1200
SAMPLE
#Channels
=lo00
0-23MA
Emitter Current
=lo93
I
I
I
I
0
200
400
600
Channel #
L 1 800
0
n
High Resolution FD vs E1
I
Hesolution =10000 # Channels 400 7 Scans
3
h v i u M ~715 1731
1
0
100
Channel
200
300
100
2QQ 300 CHANNEL #
400
500
Flgure 10. High-resolution C I molecular ion peak (M 4- H) of 4 using MSA. The reference peaks were m l z 354.9793 (on left) and 355.9826 of PFK, obtained by EI.
I
loo0
Flgure 8. Low-resolution FD spectrum of 2 uslng MSA. Tris(perfluorohepty1)trlazine was used for mass calibration.
c “QSMMU 48 23 6 0 1-26 50 25 3 1 1 -1.3 47 27 2 4 1 -40 41 27 6 5 1 32 43 29 3 6 1 45 38 29 5 8 1 05 40 31 29 1 19 33 29 7 101 -35
I I
REF
400
+t
Flgure 9. Hlgh-resolutlon FD data for molecular Ion of 3 using MSA. The correct elemental cornposition Is underllned.
a given current until no additional ions were produced. The spectrum shows a strong molecular ion at m / z 1093 with the expected isotope pattern for the chlorinated molecular ion. The fragment ion m/z 1058 represents the loss of a chlorine atom from the molecular ion. In none of the previous FD experiments had the molecular ion been seen. The loss of chlorine occurred at such low emitter currents that it was undetectable by standard techniques, but the MSA allowed the observation of such transient species by recording all ions produced even at the lowest emitter currents. High-Resolution FD. Several major problems are associated with high-resolution measurements. The first involves the great decrease in sensitivity resulting from the requirement that much of the ion beam must be attenuated to obtain a beam of uniform energy. The second is a related problem of sample lifetime. Many times it has been necessary for samples to provide a stable ion current for periods of up to several hours in order to obtain good peak matching measurements. Consequently, high-resolution FD for many samples such as those in Figures 7 and 8 has been out of the question. Another problem is the deactivation of FD emitters by materials such as PFK and Fomblin. The ability to acquire sample and reference spectra independently and in different ionization modes with the MSA and later superimpose them allows the use of these deactivating reference materials. With the development of the MSA system, many samples for which it was previously impossible to obtain high-resolution data are handled routinely. The normal procedure involves obtaining the high-resolution spectrum of the ion of interest at 10000 resolution. Generally, five to ten scans are acquired while the emitter current is increased from 0 to 23 mA. Figure 9 shows the high-resolution spectrum for the molecular ion of a derivatized nucleoside, 3, obtained by using this technique.
The reference was generated by use of the single peak method. In this case, the single reference peak chosen had a mass higher than the sample, so the second reference peak was created by increasing the accelerating voltage enough to shift the PFK peak a t m / z 715.9677 to m / z 715.1096. The high-resolution measurement gave M+ = 715.1731. Eight elemental compositions were generated that were consistent with this mass, but the expected formula, C38H29N508S,had the smallest deviation (A 0.5 mmu). High-ResolutionCI. Historically,FD was the driving force for developing the MSA technique, but other ionization modes have also been employed with the MSA. Chemical ionization has been used a great deal in the last few years to obtain molecular weight information for compounds that do not give molecular ions by EI. Like some of the other soft ionization techniques, CI tends to give large quasi-molecular ions with less fragmentation than found in E1 and it also shares another characteristic of other soft ionization techniques such as FI or FD-the general unavailability of reference standards necessary to obtain high-resolution spectra. This problem was dealt with for FD in the above examples by using a combination EI/FD source and the MSA, which allowed an E1 spectrum of the reference material to be produced by the same source as the FD spectrum. This same technique has been applied to CI using a combination EI/CI source. The use of combination ionization sources to calibrate data systems for the acquisition of high-resolution data is not ideal since the source is allowed to detune when switching modes, but this loss of sensitivity is acceptable for MSA acquisitions (9). As above, the MSA is used to acquire the accurate spectrum of the ion of interest in the CI mode, then the source is switched to E1 operation and the reference spectrum obtained. A typical example is the quasimolecular ion (M H)+ of methyl behenate (4,C23H4602,Figure 10) at m / z 355. The high-resolution measurement of the (M + H)+ ion was obtained in the CI mode with isobutane as the reagent gas. Six 5-s scans were taken using 500 channels at 10000 resolution. The source was then switched over to E1 and the reference spectrum was collected under the same conditions. The reference peaks used were m / z 354.9793 of PFK and its 13C isotope peak at m / z 355.9826. This gave a value of 355.3593 for the (M H)+ ion, agreeing with the elemental composition C23H4702 (A -1.7 mmu). As with the mixed ionization mode measurements using FD/EI on the MAT 731, no shift in the ion beam position was observed between the E1 and CI modes on the VG 7070H mass spectrometer. The above example shows the use of two different ionization modes (EI, CI) to obtain HRCI peak matching results. However, the MSA’s sensitivity is sufficient to allow PFK to be used as the reference directly in the CI mode. For this HRCI measurement, 500 channels at 10 000 resolution were used, with methane as the reagent gas. The sample, a de-
+
+
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984 C H N O MMUI 37 22 7 I 6.0 39 24 4 2 7,3 38 22 5 2 -5 3 402423-39 342464 33 3322 7 4 - 9 3 362635 46 35 24 4 5 -80 3 8 2 8 0 6 60 37 26 I 6 -6.6 31 26 5 7 0.6 332828 1 9
High Resolution CI (Methane) vs CI Resolution: 10000 # Channels 500,
4'Sca;ls
n 14 127
Table 11. MSA Reproducibility
I
0
Table I. MSA Measurement Accuracy av error, std dev PPm mode 4.94 5.53 FD 4.87 4.20 FAB
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100
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I
I
I
200
300
400
500
compound
av calcd mass,amu
erythromycin
756.4487
methanodidemnin
955.5698
stddev
n
2.04 mmu 2.70 ppm 1.24 rnmu 1.30 ppm
6
3
CHANNEL #
+ H) of 5 using MSA. The correct elemental composition Is underlined. The references were PFK peaks. Flgure 11. High-resolution CI molecular ion peak (M
0
100
200
300
400
500
CHANNEL NO.
Flgure 12. High-resolution FAB molecular ion (M - H 4- 2Na) of streptolydigin, 8 (peak B, 645.2779), obtained by using MSA.
rivatized nucleoside (5, C31H2BN507, Figure 111,required four 5-s scans. While the sample spectrum was being obtained, the PFK reference was introduced through the reference inlet system. After the data for the (M + H)' ion (m/z 580) had been collected, a 30-fold vertical expansion showed two additional peaks (Figure ll),the PFK reference peaks at m/z 579.9744 and m/z 580.9633. Using these peaks as references yielded a value of 580.1826 for the (M + H)' ion. Of the 12 elemental compositions consistent with this result, the correct formula, C31H26NS07, had a deviation of 0.6 mmu. High-Resolution FAB. When the MSA was used in acquiring high-resolution FAB spectra, the sample spectrum is obtained in the FAB mode at 10 000 resolution and the reference spectrum in either the FAB or E1 mode. For the measurement shown in Figure 12 ten 6.8-s scans were obtained with 500 channels to give a high-resolution FAB ion of streptolydigin (6, Figure 12) superimposed on a high-resolution E1 ion of the reference. Peak A represents m/z 644.9429 of Ultramark 1600F while peak C was generated by shifting peak A by 1400 ppm to 645.8458. The quasi-molecular ion (M H + 2Na) of streptolydigin (peak B) was measured a t m / z 645.2779 (A 1.5 mmu). High-resolution FD and FAB experiments have constituted the major work load for the MSA in our laboratory. Table I summarizes accuracy data from measurements made over the past 2 years. The MSA has been able to provide routinely high accuracy for FD and FAB mass measurements, which
is particularly noteworthy since some of the FD measurements would have been otherwise impossible due to poor SIN and/or lack of useful reference materials. Measurements made with the MSA are also very reproducible, as indicated by the precision data from some FAB experiments presented in Table
11. Isotope Ratios. There are many applications for isotope ratio determinations, such as studies of the abundance of the natural isotopes (19) and the incorporation of isotopically labeled precursors in a biosynthetic pathway (20, 21), and specialized mass spectrometric techniques are usually employed for these determinations, including use of mass spectrometers equipped with multiple detectors (20,22). The conventional method for carrying out isotope ratio measurements on an instrument with a single detector involves slowly scanning the mass range of interest and recording the peaks oscillographically (20). The heights, or preferably the areas, of these peaks are then used to determine the isotope ratio. Ideally, the measurement should be carried out at sufficient resolution to separate all peaks containing isotopes, but this is not always possible, since the required resolution is beyond the capabilities of all but a few ultrahigh resolution instruments and the loss of sensitivity at the required resolution is severe. Because of the problems associated with high resolution, the normal procedure when a single isotope is involved requires running the spectrum at modest resolution and subtracting the contribution of unlabeled material (23). Data obtained on a sample of the unlabeled material are used to determine the natural isotopic abundance of the ion of interest, and the labeled sample is then run under identical conditions. The isotopic contribution from the unlabeled sample is subtracted, and this corrected value is used to calculate the isotopic enrichment. Isotope ratio measurements using the oscillographic procedure are easily obtained from a heated inlet system and with only slight difficulty from a direct probe since the amount of sample entering the mass spectrometer per unit time can be maintahed reasonably constant. However, when isotope ratios must be measured from a GC peak, the sample concentration as well as the isotope ratio is changing with time. One solution to this prdblem of changing sample concentration involves scanning at the top of the GC peak where the concentration of sample is relatively constant. Unfortunately, this region of the peak is rather narrow: for a typical capillary GC peak 6 s wide, the region of relatively constant Concentration at the apex may be only 1to 2 s wide. Even if the operator can spot the apex exactly, no more than one or two scans can be acquired, not enough to satisfy statistical considerations. Another solution is to acquire and average multiple scans across the entire GC peak. This method effectively removes the effect of time on the sample concentration by integrating
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984 101
2039
8C
1
3
7
6C
X I
uc 81
2C
j1 1
3
a 50
IT1
272
189 2 9
149 I
I,
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I,,,
jlp~
,11
1'50
f,
2
,,
1
7
250
Flgure 13. Low-resolutlon E1 mass spectrum of unlabeled casbene, 7.
1
0
Table 111. Natural Abundance Contributions to the (M + 1 ) Peak of an Unlabeled Sample of Casbene run no. 1 2 3 4 5 6 7
CHANNEL #
peak areas M (M t 1) (M 1325389 1617457 1365900 2277349 2287478 2572040 2403686
284725 353907 278910 464949 447251 506138 486951
av
f i b
run
the entire area under the peak but requires the acquisition of a large number of scans over a short period of time. The need for a fast, reproducible method of scanning over a limited mass range is perfectly suited to the MSA approach, which affords two advantages over the oscillographic method. First, since the MSA is capable of a scan time of 0.3 s, approximately 30 scans can be obtained for a typical 10-s GC peak. Second, the MSA sums the scans as they are acquired, keeping a running total of the peak area. In contrast to the many feet of oscillographic paper which must be measured by hand at the end of an experiment, a single report, with a trace of the
100%
21.48 21.88 20.42 20.42 19.55 19.68 20.26 20.53 f 0.64
Table IV. Incorporation of Deuterium into Casbene, Calculated from (M + 1 ) Peak Areas for Sample 5-37-RUsing MSA no.
Flgure 14. Isotope ratio GC/MS using MSA: (a) molecular Ion region of unlabeled casbene, 7; (b) same reglon of monodeuterated casbene.
+ 1)/M x
1 2 3 4 5 av
normalized peak areas M (M t 1) 100.00 100.00 100.00 100.00 100.00
118.90 120.22 120.80 119.30 120.51
%
incorporation 49.60 49.92 50.07 49.69 50.00 49.86 f 0.18
peaks scanned and their areas, is produced by the MSA. Figure 13 contains the E1 mass spectrum of casbene (7, C20H32)which can be labeled with deuterium (24) by incubation of the monodeuterated precursor with a crude enzyme extract. Because the isolation of the compound from the reaction mixture is difficult, the sample was submitted for isotope ratio measurement by the GC/MS technique. The MSA was set up to run 0.3-s scans with 100 channels over the mass range m / z 272 to m / z 274 (Figure 14). The scanning was begun 3 min and 25 s after injection to avoid interference by background peaks in this region.
Anal. Chem. 1984, 56, 1481-1487
Seven separate GC/MS determinations were made with unlabeled casbene (Table 111), with each run representing 30-40 s. An average value of 20.53% was obtained for the ratio of (M 1)/M for unlabeled casbene us. a theoretical value of 22.48%. Table IV contains the results of five runs using deuterium labeled casbene from the enzyme preparation. After subtracting the previously determined I3C contribution of unlabeled material (20.53%) from the M + 1ion intensity of the labeled material, we found an average value of 49.86% incorporation. This value is only slightly higher than previous results obtained by using the oscillographic technique (49.1%). A different enzymatic preparation of labeled casbene gave an incorporation of 50.60 f 0.85%. Within experimental error, these two results are identical and are well within the 1-5% error generally associated with this type of isotope measurement (25).
+
LITERATURE CITED Kennett, B. H. Anal. Chem. 1987, 3 9 , 1506. Blros, F. J. Anal. Chem. 1970, 5 2 , 5357. Peele, 0. L.; Brent, D. A. Biomed. Mass Spectrom. 1978, 5 , 180. Hass, J. R.; Friesen, M. D.; Harvan, D. J.; Parker, C. E. Anal. Chem. 1978, 5 0 , 1474. Van Ness, G. F.; Solch, J. 0.; Taylor, M. L.; Tiernan, T. 0. Chemosphere 1980, 9 , 553. Lenmann, W. D.; Schulten, H. R. Angew. Chem., Int. Ed. Engl. 1877. 16, 184. Ligon, W. V., Jr. Abstracts, 27th Annual Conference on Mass Spectrometry and Allied Topics, Seattle, WA, June 3-8, 1979; p 481. Ligon, W. V., Jr. Abstracts, 28th Annual Conference on Mass Spectrometry and Allied Topics, New York, May 25-30, 1980; p 490. Snelllng, C. R., Jr.; Cook, J. C., Jr.; Milberg, R. M.: Rlnehart, K. L., Jr. Abstracts, 29th Annual Conference on Mass Spectrometry and Aliled
1481
Topics, Minneapolis, MN, May 24-29, 1981; p 602. (10) Savitzky, A.; Goiay, M. J. E. Anal. Chem. 1964, 36, 4527.
(11) Snelling, C. R., Jr; Milberg, R. M.; Cook, J. C., Jr.; Rinehart, K. L., Jr. Abstracts, 28th Annual Conference on Mass Spectrometry and Aiiied Topics, New York, May 25-30, 1980; p 484. (12) Ligon, W. V., Jr. Int. J. Mass Spectrom. Ion Phys. 1982, 41, 213. (13) Mueller, P.; Rudin, D. 0. Nature (London) 1968, 217, 713. (14) Roy, G. J. Membr. Biol. 1975, 2 4 , 71. (15) Symposium on Membrane Channels Fed. R o c . , fed. Am. Soc. Exp. Biol. 1978, 37, 2626. (16) Rinehart, K. L., Jr.; Pandey, R. C.; Moore, M. L.; Tarbox, S. R.; Snelling, C. R.; Cook. J. C.. Jr.; Milberg, R. H. I n “Peptides: Structures and Biological Functlon 1979”; Gross, E.; Meienhofer, J., Eds.; Plerce Chemical Co.: Rockford, I L 1979; pp 59-71. (17) Rlnehart, K. L., Jr.; Gaudioso. L. A.; Moore, M. L.; Pandey. R. C.; Cook, J. C., Jr.; Barber, M.; Sedgwick, R. D.; Bordoli, R. S.; Tyler, A. N.; Green, B. N. J. Am. Chem. SOC.1981, 103, 6517. (18) Rlnehart, K. L., Jr.; Cook, J. C., Jr.; Meng, H.; Olson, K. L.; Pandey, R. C. Nature (London) 1977, 269, 832. (19) Beynon, J. H. “Mass Spectrometry and Its Applications to Organic Chemistry”; Elsevier: New York, 1960; pp 294-302. (20) Caprioli, R. M. “Biomedical Applications of Mass Spectrometry”; Waller, G. R., Ed.; Wliey-Interscience: New York, 1972; pp 735-776. (21) Caprloll, R. M.; Bler, D. M. “Blomedlcal Appiicatlons of Mass Spectrometry, First Supplementary Volume”; Wailer, G. R., Dermer, 0. C., Eds.; Wiley-Interscience: New York, 1980; pp 895-925. (22) Reference 19, pp 201-203. (23) Millard, B. J. “Quantltatlve Mass Spectrometry 1979”; Heyden: London 1979; pp 62-66. (24) Robinson, D. R.; West, C. A. Blochemistry 1970, 9, 70. (25) Reference 19, p 100.
RECEIVED for review December 28,1983. Accepted March 26, 1984, This work was supported in part by a grant from the National Institute of General Medical Sciences (GM27029). This report is taken in part from the Ph.D. thesis of C. R. Snelling, Jr., University of Illinois, Urbana, 1981.
Supercritical Fluids as Spectroscopic Solvents for Thermooptical Absorption Measurements R. A. Leach and J. M. Harris* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
Dense gases near the liquid-vapor critical point are shown to possess advantageous properties for use as solvents In thermoopticai absorption measurements. The sensltivlties of thermal lens and photothermal deflection spectroscopies have been studied in carbon dioxide near its crlticai point as a function of temperature and density. A 100-fold sensitivity Increase, relative to measurements in carbon tetrachloride, has been achieved for both thermoopticai methods tested. The theory of critical point sensitivity enhancement is developed by using P-Y-T data on C02 from the literature. Experimental results obtained are found to closely follow theoretical predictions.
A new class of sensitive spectrophotometric techniques has been developed in which the heat produced by nonradiative decay of excited species acts to modify the optical properties of the sample (I). These thermooptical absorption techniques can be classified by their spatial temperature distribution and corresponding refractive index change, which determines the particular method of detecting the heat produced in the sample. For example, if the sample if uniformly heated by a large-area excitation beam, the change in optical path can be detected interferometrically (2). When a focused laser 0003-2700/84/0356-1481$01.50/0
beam is used for excitation, the resulting temperature gradient produces a lenslike optical element which can be measured by its effect on the divergence of the laser beam (3,4). A periodic temperature distribution can be generated by an excitation interference pattern and probed as a thermal transmission grating by diffraction of a laser beam into a detector (5,6). Finally, by creating a thermal gradient in a sample with optical excitation at an interface, the resulting thermal prism can be detected by its deflection of a laser beam (7). Unlike conventional transmission or reflection measurements, the sensitivity of these thermooptical methods depends on the power of the radiation used for excitation and the thermophysical properties of the sample. Solvents which exhibit a large change in refractive index with temperature, dn/dT, are advantageous since a given increase in temperature produces a large change in optical path. For continuous wave (CW) excitation, solvents of smaller thermal conductivity produce larger temperature gradients at steady state, where the rate of heating is balanced by the rate of thermal diffusion. Supercritical fluids or dense gases are a class of solvents which are generating considerable interest and applications in analytical chemistry. As mobile phases for chromatography, supercritical fluids provide greater chromatographic efficiency than normal liquids due to their larger diffusion coefficients, 0 1984 American Chemical Society
