Anal. Chem. 1982, 5 4 , 332-334
332
Exact Mass Measurements with a Computer-Controlled Peak Matching System D. J. Harvan," J. R. Hass, and D. Wood' National Instltute of Environmental Health Sciences, P.0. Box 12233, Research Triangle Park, North Carolina 27709
The determination of the elemental composition of an unknown molecule provides one of the most valuable pieces of information available for its identification. In general, exact mass measurements using a high-resolution mass spectrometer enable the assignment of the molecular formula (1). When high-resolution mass spectrometry (HRMS) is used in conjunction with high-resolution gas chromatography (HRGC) for the analysis of complex mixtures, a number of limitations become apparent (2). At a given scan rate, the time width of the mass spectral peaks decreases until some limit is reached by the signal amplifiers. Given sufficiently fast scanning of the magnetic field, this limit is presently approximately 2-3 s/decade of mass a t 8000-10000 resolving power. Thus, scanning a complete high-resolution mass spectrum may require 5-10 s. This is approximately the same as the full elution time of a typical HRGC peak. Resolutions greater than loo00 are thus not practical. A second problem is that fewer ions actually arrive at the collector as one increases the scan speed, resulting in a loss of sensitivity. This is compounded by the reduction in ion beam transmission with increasing resolution which results in further losses in sensitivity. For reasonably pure samples (i.e., a homogeneous GC peak) the effects of these problems can be reduced by using lower resolution (1000-2000) and a reference compound with a relatively small number of types of ions which have large negative mass defects ( 3 , 4 ) . This method usually gives acceptable results on the mass assignment of the molecular ion. Difficulties are experienced in the mass assigment of fragment ions when doublets occur as a result of isobaric losses (Le., M - CO and M - CzH4 would normally not be resolved) or a t low sample levels when instrumental background and column bleed may significantly shift peak centroids. Naturally, if a coeluting interference is present, assignment errors will occur. Typically, in HRGC analysis using this technique, most elemental compositions can be assigned. A limited number of specific questions will remain which require either higher resolution and/or greater sensitivity. In this paper, we describe a method for acquiring limited mass range high-resolution mass spectral data without significant loss in chromatographic resolution and with acceptable sensitivity for most analyses of environmental samples. The method we have employed is computer-controlled peak matching with full peak profiie information being stored. Computer-controlled peak switching has been previously described, but peak profile information was not obtained (5-10). The method requires only minor modifications to commercially available equipment.
EXPERIMENTAL SECTION Mass spectral data were acquired with a VG Micromass W - 2 F spectrometer, a modified multiple ion detection (MID) unit, and a modified Finnigan-Incos 2300 data system. Normally, the MID unit allows high-resolution peak matching of up to seven masses relative to a standard "lock mass". The MID unit samples the channel containing the lock mass within a narrow scan window (0-1000 ppm) and makes 5 ppm corrections of the accelerating voltage if the peak is not centered in the window. The accelerating voltage is then programmed via a peak matching unit to transmit ions of a selected exact mass. Present address: VG Instruments, 300 Broad St., Stamford,CT
06901.
0003-2700/82/0354-0332$01.25/0
Two modifications have been made to the MID unit (Figure 1): (1)the unit will cycle through the MID program only upon receiving a start-of-scan pulse from the data system (section A) and (2) the 30-mS blanking pulse between channels (to allow settling of the accelerating voltage) has been inverted and added to the amplifier output (section C). The first modification allows the data to be synchronized so that multiple scans can be summed by the data system and thus improve the ion statistics of the acquired data. The second modification allows the limits of the scan windows to be marked. The data system calibrates the X axis as if it were linear mass scan. The scan is actually a linear mass scan with discontinuities between channels and is converted into a corrected mass axis by the operator. Therefore, if the operator adjusts the MID system for "X" ppm scan and the data system records the mass between the channel markers as "Y" amu, then the window is calibrated in ( X / Y ) ppm/"amu". The exact mass of the center of the window is known because it is set by the operator relative to the lock mass. Thus, any mass peak that occurs in the window can be mass measured by the number of parts per million it deviates from the center of the window. The interface box of the data system has been modified in two ways: (1)the 400 ns start-of-scanpulse from the digital transceiver printed circuit board is used to close the relay which triggers the MID unit (section B)and (2) the digital transceiver card has been modified to allow the user to select the desired analog-to-digital conversion frequency. The first modification allows electronic isolation of the mass spectrometer from the data system. The second modification allows slower acquisition rates to accommodate the slower mass scans employed (500 ppm/0.5 s = 1000 ppm/s = 0.3 amu/s at mass 300).
RESULTS AND DISCUSSION Figure 2 shows the peak profiles at 40 000 mass resolution for the m / z 154.9920 (C6F5),161.9904 (C4F6),and 162.9983 (C4F6H)ions of perfluorokerosene. The m/z 163 channel has small signals at m/z 162.9332 (?) and 162.9938 (13CC3F6).The 155 ion was the lock mass for the MID unit and the channel span was adjusted for 550 ppm. The signal a t m / z 162 was found to deviate by 6 ppm from the center of the channel, its exact mass being measured as 161.9914. The ions in the m / z 163 channel were measured as 162.9342 (measured as 162.9332 by manual peak matching; 4.3 ppm error), 162.9945 (4.3 ppm error), and 162.9990 (4.3 ppm error). Also monitored in this experiment was the m / z 180.9888 ion, and its centroid was measured a t 180.9904 (8.7 ppm error). The data in Figure 2 represent the summation of nine scans. The ability to resolve the 13C-12CH doublet (required resolution = 36000) demonstrates that the system is sufficiently stable to not degrade the mass spectrometric resolution by signal summation. The accuracy of measuring the exact mass of components eluting from a capillary column (OV-1 25 m, fused silica) was evaluated by monitoring a 300 ppm window at rn/z 142.9920 (C4F5,lock mass), 149.0238 (C8H603),and 178.0783 (C14H11)) at 10 000 resolving power for a mixture containing diethyl phthalate (DEP), di-n-butyl phthalate (DBP), a diethylhexyl phthalate (DEHP), and anthracene (Anth). The lock mass was monitored for 0.2 s per scan, and the m / z 149 and 178 ions for 1s each, with a total scan cycle of 2.4 s. The resulting GC trace (Figure 3) illustrates the signals observed for approximately 10 pg of each component. The deviation of the mass assignments as a function of amount of sample injected on-column is shown in Table I. Duplicate runs are reported for the two concentrations. The signal for DEHP a t 10 pg 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
333
Table I. Mass Measurement Accuracy as a Function of Sample Size error, ppm compd
GC scan no.
DEP DEP DEP REP DBP DBP DBP DBP DEHP DEHP DEHP Anth Anth Anth Anth
115 116 116-117 113-115 214-215 215-217 215-217 213-215 407 407-409 403-406 168-170 169-171 170-173 167-170
10 pg each 149.0238 178.0783
100 pg each 149.0238 178.0783
-7.6 6.5 -1 3.2 -1.8 0.7
-1 0.7 3.2 2.4 -1.8 -4.4 -3.6 1.4 0.6
2E51
Y T MIX I O p g
A
149
I 1 t o MID channel cycle
ZES
E
L
1170
1
ANTH
t 14
C
to data
reset
I--------
from amplifier
UNIT
--
I
I
PFK 4 0 K R P
C4FE
INT
,
550
161.9904
,,
200
3'00
400
8:oo
12.00
16.00
- Tirne(mln)Figure 3. GC trace for ( m l z 149) phthalates and ( m l z 178) anthracene at 10 000 mass resolution. The TIC also includes the ion current from PFK m l z 143 ion as lock mass.
injected was not detected for one of the trials and was at a
OEI
154.992
IbO 4 00
S I N of 2.5:l for the other measurement. The S I N was approximately 1O:l for the other phthalates and 25:l for an-
Figure 1. Modifications to interface and MID unl. (A) Start scan pulse from digital transceiver used to drive relay, thus actlvating (B) the MID channel cycle. The MID channel cycle relay is then reset by the scan reset of the oscilloscope. The switch allows manual operation of MID channel cycling. (C) The channel reset pulse is inverted and added to the amplifler signal to mark the steps between channels.
m/z
1
5
i
span
D
162.9653
ppm-
Figure 2. Perfluorokeroseneat 40 000 resolution. The channel marks are seen as small pulses on the base line.
thracene for the 10-pg injections. Similar results were obtained at 45 000 resolving power and a 400 ppm scan window. The mass assignments were accurate to 2 ppm for 2 ng injected on-column. In general, we have been able to measure exact masses with an accuracy of f 5 ppm with resolutions from 10 to 50000 and scan windows up to 600 ppm. In the current instrument the lock mass correction of 5 ppm leads to an unacceptably large deviation in mass assignments and will be reduced in later versions. A second difficulty is encountered if one rapidly scans a large window at higher resolutions. Insufficient time is spent on the lock mass peak for proper sampling and thus no correction is applied. However, one has the data system mass assignment of the lock mass centroid so that any drift can be corrected after data acquisition. A third difficulty is that the peak matching unit employs precision resistors with temperature specifications of f 3 ppm "C, and the fluctuations in room temperature may introduce some error. Also, a slope error is inherent in the use of a peak-matching system. As peak-matching ratios become larger (>l.l), errors may range up to 10 ppm. These limitations arise from the use of MID and peak matching units. The use of a precision digital-to-analog converted and development of appropriate software to incorporate a lock mass capability within the data system will
334
Anal. Chem. 1982, 5 4 , 334-336
circumvent some of these difficulties. We are in the process of developing such a system.
ACKNOWLEDGMENT The authors thank John Sadler (VG Analytical) for design of the MID circuitry, Ben Johnson (Finnigan-MAT) for modifications to the interface to the data system, and Mark Weiss (Finnigan-MAT) for helpful software discussions.
LITERATURE CITED (1) Beynon, J. H. “Mass Spectrometry and Its Application to Organic Chemistry”; Eisevier; Amsterdam, 1960;pp 312. (2) Kimble, B. J. I n “High Performance Mass Spectrometry: Chemical Applications”; Gross, M. L., Ed.; American Chemical Society: Washington, DC, 1978;Chapter 7; ACS Symp. Ser. No. 70. (3) Hadden, W. F.; Lukens, H. C. Presented at the 22nd Annual Confer-
ence on Mass Spectrometry and Allied Topics, Philadelphia, PA, 1974,
u-2.
(4) Craig, R. D.; Baternan, R. H.; Green, B. N.; Millington. D. S. Ph//os. Trans. R. Soc. London, Ser. A 1079, 283, 135-155. (5) Hammar, C. G.; Hessiing, R. Anal. Chem. 1071, 43, 298-306. (8) Hammar, C. G.; Pettersson, G.; Carpenter, P. T. B/omed. Mass Specfrom. 1074, 1, 397-411. (7) Seller, N; Knoedgen, B. Org. Mass Spectrom. 1973, 7. 97-105. (8) Meredith, J. 0.; Southon, F. C.; Barber, R. C.; Williams, P.; Duckworth. H. E. Int. J . Mass Spectrom. Ion Phys. 1073, 10, 359-370. (9) Gruenke, L. D.; Cymerman, C. J.; Bier, D. M. B/omed. Mass Spectrom. 1074, I, 418-422. (10) Hadden, W. F. I n “High Performance Mass Spectrometry: Chemlcai Applications”; Gross, M. L., Ed.; American Chemical Society: Washington, DC, 1978,Chapter 6;ACS Symp. Ser. No. 70.
RECEIVED for review August 31, 1981. Accepted October 23, 1981.
Microelectrolytic Cell for Voltammetric Analysis Joseph Wang’ and Bassam A. Freiha Department of Chemistty, New Mexico State UniversiW, Las Cruces, New Mexico 88003
It has been demonstrated in the literature that electroanalytical techniques can be adapted to analyses of very small sample volumes. Most electrochemical cells with small volume capability are based on the thin-layer principle (usually in a “sandwich type” configuration) ( I , 2). Other cells or approaches include a rotating disk electrode microcell (3, 4 ) , capillary packed-bed electrodes (5),mercury drop electrode microcells (6),specially designed cells with stationary carbon electrode (9, as well as micro flow cells for liquid chromatography and flow injection analysis (8). Thin-layer cells suffer from problems such as “iR distortion” or “edge effect” (9, IO), and they are relatively expensive and difficult to construct. With the growing need for sensitive and reliable analyses in microliter sample volumes it becomes necessary to explore other microcell configurations. We report here a very simple and inexpensive microelectrolytic cell. The cell utilizes a drop of the test solution placed on an inverted carbon paste disk electrode, with a capillary reference electrode immersed in the drop. The sample solution defines its geometry over the electrode via surface tension. A cell of similar philosophy, but of different geometry, has been described (7). However, little has been reported concerning the response characteristics of this cell. Advantageous features of the present cell include simplicity and low cost of construction, ease of use, and sensitive and reproducible response. Thus the cell minimizes some of the disadvantages of thin-layer cells. Measurements are performed on 50 p L solution volume. Differential pulse voltammetry (DPV) is employed for measuring dopamine, chlorpromazine, and ferrocyanide ion at the micromolar concentration level. EXPERIMENTAL SECTION The cell design is shown in Figure 1. The body consisted of two 3.5 X 3.0 X 1.2 cm Plexiglas blocks, held together with two stainless steel bolts (at opposite corners). A 0.3 cm diameter hole was drilled in the center of the lower block to accommodate the carbon paste working electrode. The paste was made by thoroughly mixing graphite powder (Acheson Graphite, Grade 38, Fisher) and Dow Corning silicone grease (43% silicone grease by weight). The paste surface was smoothed using a computer card, and ita other end was connected to a copper wire (epoxied in the hole) that led to the outside. The geometric area of the working electrode is approximately 0.07 cm2. A 50-pL sample drop was placed on the carbon disk, covering all the surface of the electrode. A hole (0.5 cm widened to 1.5 cm diameter) was drilled in the center of the upper block to contain the reference electrode and protect the sample against contamination and evaporation effects
(such protection was not provided in the cell described in ref 7). The Ag/AgCl reference electrode was made of a capillary tube (Kimble Products, Toledo, OH) of 1.8 mm o.d., which contained a thin silver wire and 0.1 M KC1 solution. The reference electrode was immersed in the solution drop, about 2 mm above the center of the carbon paste disk (the close proximity of the two electrodes minimized distortion effects due to ohmic polarization). Samples were removed by washing the electrodes with deionized water, followed by drying the working electrode with a stream of air. A 50-pL Hamilton microsyringe was used to place new samples. In all experiments, a Princeton Applied Research Model 364 polarographic analyzer was used with a Houston Omniscribe chart recorder. Chemicals and reagents used have been described previously (11) excepted as noted. Chlorpromazine hydrochloride was obtained from Sigma Chemical Co. All solutions were prepared daily by weight in 0.1 M phosphate buffer, pH 7.4.
RESULTS AND DISCUSSION Figure 2 shows linear scan and differential pulse voltammorgrams for the oxidation of 10 FM dopamine and 100 HM chlorpromazine in pH 7.4 phosphate buffer. The voltammograms are well-defined (and reproducible) and the background current is low. The DPV peak potentials for dopamine and chlorpromazine are a t +0.08 and +0.8 V, with widths a t half-height of 93 and 210 mV, respectively. Voltammograms with similar potential regions and shapes have been observed for dopamine and chlorpromazine at various carbon electrodes (12,13). The dopamine linear scan response exhibits a nearly steady-state plateau rather than diffusion limited current decay. This behavior may be attributed to radial diffusion to the edges of the disk, as will be discussed later in the paper. The advantages of DPV, over linear scan voltammetry, for determining low concentrations of easily oxidized organic molecules are obvious. Detection limits at the submicromolar concentration level are predicted from the signal to noise characteristics of the DPV data. The degree of carry-over, i.e., the influence of the concentration of one solution on the result obtained for the subsequent solution, may be tested by sequential determinations of sample and blank solutions. Results of such a test run with 50 pM dopamine solution followed by ita corresponding blank (0.1 M phosphate buffer) solution are shown in Figure 3. Well-defied peaks (with similar peak heights-4.99,0.98, and 1.01 pA) are obtained, with no observable carry-over between the sample and blank solutions. The sample replacement procedure (washing, drying, etc.), described in the Experi-
0003-2700/82/0354-0334$01.25/00 1982 American Chemical Society
