Anal. Chem. 1991, 63,713-721
713
Collision Energy, Collision Gas, and Collision Gas Pressure Effects on the Formation of 2,3,7,8-Tetrachlorodibenzo-p -dioxin and 2,3,7,8-Tetrabromodibenzo-p -dioxin Product Ions M. Judith Charles* and G. Dean Marbury Department of Environmental Sciences and Engineering, University of North Carolina a t Chapel Hill, Chapel Hill, North Carolina 27599-7400
The effects of collision energy, the nature of the collision gas, and collision gas pressure on formation of product Ions of 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrabromlnated dibenzo-p-dloxin (TBDD) and approaches to determine optimum conditions for mass spectrometry/mass spectrometry (MSIMS) of TCDDs and TBDDs were Investigated. The results demonstrate the optlmum coillslon energy conditions are dependent on the pressure of argon, nitrogen, and xenon and independent of helium pressure. For similar pressures, the optimum collision energy for formation of the (M - COCI)’ and (M - COB$ product ions was lowest for helium and xenon (6-10 eV) and about the same for argon and nltrogen (20-30 eV). Shnliar optima were observed when argon or nitrogen were used as coilision gases, thus indicating the importance of cdlision diameter and mean free path in productlon of TCDD and TBDD product Ions. Because of the relationship between collision energy and collision gas pressure, a systematic approach to optimization experiments In which the collision energy is varled at specific pressures is suggested. Detectlon sensitivities (signal:noise, S:N) for 1 pg of 2,3,7,8-TCDD under optimal condltlons are 2:l for He, 7:l for Ar, 9:l for N,, and 1O:l for Xe. Detectlon sensltivltles for TBDD relative to TCDD are a consequence of greater fragmentation and lower detection sensltivlties of the first mass analyzer. A S:N of 12:l for He and At, 7:l for N,, and 11:l for Xe was measured on 100 pg of TBDD.
INTRODUCTION The use of mass spectrometry/mass spectrometry (MS/ MS) for the analysis of trace levels (ppb, ppt) of tetrachlorinated dibenzo-p-dioxins (TCDDs) in environmental samples is increasing because in certain cases MS/MS is the only technique that can separate molecular or fragment ions of compounds (e.g., polychlorinated biphenyls, alkylated dibenzofurans) from the molecular ion of TCDD. In these situations, chromatographic fractionation procedures and high-resolution mass spectrometry (resolving power = 10OOO and 18000) do not provide the selectivity that can be obtained by selected-reaction monitoring (SRM) of the (M - COCl)+ product ion (I, 2, 3 ) . The efficiency of formation of product ions in MS/MS depends on the collision energy, nature of the collision gas, and collision gas pressure ( 4 , 5). The importance of these parameters can be understood by examination of the center of mass collision energy equation:
Ecom= &b(mg/(mp + mg)) where E,,, is the center of mass collision energy which equals the maximum translational energy available for conversion to internal energy, Elab is the energy in the laboratory frame 0003-2700/91/0363-0713$02.50/0
or collision energy, mg is the mass of collision gas molecule, mp is the mass of parent ion. The maximum translational energy available for internal conversion increases by raising the collision energy (Elab) and using heavier target gases. Increasing the gas pressure increases the number of ions produced under single-collision conditions, and increasing the energy of deposition into the parent ion causes more fragmentation under multiple-collision conditions. In general, greater production of product ions occurs as the collision energy, mass of collision gas, and collision gas pressure are increased ( 4 ) . Accordingly, information on the effect on collision energy, the nature of the collision gas, and collision gas pressure on formation of product ions of the analyte is needed to determine optimum conditions for analysis. Several approaches have been used to determine these conditions by using argon as the collision gas in MS/MS analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs, PCDFs). In one approach, the pressure was chosen by attenuating the main beam of perfluorokerosene (PFK) to 50% (1,6). This approach provides a rapid means of determining pressure conditions, independent of instrument type, but the resulting pressure may not be optimum for formation of the specific product ion of interest. An iterative approach was used by other investigators in which the optimal collision gas pressure was determined at an arbitrary selected collision energy and then the optimal collision gas energy was determined at the “optimal” collision gas pressure (7). In our laboratory, we have taken a more systematic approach. Collision energies were varied under specified pressures (2) so that the optimum conditions could be chosen by using response vs collision energy plots for different pressures. This approach has the disadvantage of being more tedious and time-consuming but the advantage of providing information about the synergistic effects between collision energy and collision gas pressure. In order to compare the results of optimization experiments conducted in different laboratories or on different types of tandem mass spectrometers, a standard approach is needed (8).
In this study, our objectives were to examine the effect of collision energy, collision gas pressure, and the nature of the collision gas on collision-induced dissociations of TCDD and tetrabrominated dibenzo-p-dioxin (TBDD) to determine optimum conditions of analysis, and to describe an experimental approach to optimization experiments that can be used in future development of MS/MS methods. EXPERIMENTAL SECTION Standard Preparation. Standards of 1,3,6,&tetrachlorinated dibenzo-p-dioxin(TCDD), 2,3,7,8-TCDD, [13C12]-2,3,7,8-TCDD, 2,3,7&tetrabrominated dibenzo-p-dioxin (TBDD), and [13C,z]2,3,7,8-TBDD were obtained from Northrop Services Inc. Standard mixtures were prepared in toluene of 1,3,6,8-TCDD, 2,3,7,8-TCDD,and 2,3,7,8-TBDDranging in concentrationsfrom 50 to 100 pg/pL 1,3,6,8-TCDD, 100 to 200 pg/pL 2,3,7,8-TCDD, 0 1991 American Chemical Society
714
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
and 500 pg/pL to 5 ng/pL 2,3,7,8-TBDD. The concentration of the solutions for a particular experiment was chosen to provide reasonable signals at 10-50-eV collision energies at a given reference pressure. Other standard mixtures of 2,3,7,8-TCDD, [13C12]-2,3,7,8-TCDD, 2,3,7,8-TBDD, and [13C12]-2,3,7,8-TBDD were prepared in toluene and used to determine the detection sensitivities of 2,3,7,8-TCDD and 2,3,7,8-TBDD under optimal collision gas pressure and collision energy conditions for a specific collision-gas. High-Resolution Gas Chromatography (HRGC). Experiments were conducted by using either DB-5 30- or 60-m 0.25 mm i.d. fused-silica capillary columns. The DB-5 60-m column was used to measure detection sensitivities (S:N measurements) for 2,3,7,8-TCDD. For analyses conducted on the DB-5 30-m column the GC oven was held at an initial temperature of 130 "C for 1 min and then temperature-programmed at a rate of 10 "C/min to 300 "C. Analyses on the DB-5 60-m column were conducted with the GC oven held at an initial temperature of 130 "C for 1 min, then temperature-programmed at a rate of 20 "C/min until a temperature of 190 "C followed by a rate of 3 "C/min to 270 "C. The detection sensitivity for TBDD under optimum conditions for a specific gas were conducted by using on-column injections. All other analyses were conducted by using split/ splitless injections. An autosampler was used for injections in the optimization experiments to reduce variability in injection error. Volumes of standard solutions injected were 1 pL, corresponding to total amounts on the order of 50 pg of 1,3,6,8-TCDD, 100 pg of 2,3,7,8-TCDD, and 1 ng of TBDD. Mass Spectrometry/Mass Spectrometry (MS/MS). Chemical standards were analyzed by HRGC/MS/MS on a Hewlett Packard 5890A gas chromatograph interfaced to a VG 70-250 SEQ hybrid mass spectrometer. The first mass analyzer (MS1) is a Nier-Johnson forward geometry double-focusing configuration (EB), followed by a collision region formed by a radio frequency (RF) quadrupole field (61) and quadrupole mass filter (Q2) as the second mass analyzer (MS2). A high-sensitivity electron ionization (EI) only source, as supplied by the manufacturer was used at a source temperature of 250 "C, electron energy of 34 eV, 5OO-crA current, and 8-kV accelerating voltage. Prior to initiation of an experiment the collision cell voltage and analyzer ion energies were zeroed by adjusting the voltage on the cell or analyzer so that single-ion signals were just observable on the oscilloscope when one of these variables was set to zero relative to the MS1 accelerating voltage of 8 kV. The first mass analyzer was tuned by using perfluorokerosene (PFK). Tuning of the second mass analyzer was achieved by first admitting either 2,3,7,8-TCDD or 2,3,7,8-TBDD into the source via the directinsertion probe and then adjusting the lenses to maximize the signal of the parent ion. Tuning on these compounds, as opposed to PFK, has been shown to enhance the signal strength (7, 9). The transmission and detection of ions through the second mass analyzer depends on the energy of the ions. The ion energy potentials were therefore adjusted for optimum transmission of the parent and product ions by adjusting the low-mass ion energy (offset) to obtain the maximum response on the (M - COCV and (M - COBr)' ions, and then setting the high-mass ion energy (slope) to obtain the maximum response on the M + 2 TCDD or the M + 4 TBDD ions. MS/MS Selected-Ion-Monitoring Experiments. The compounds were introduced via the gas chromatograph in the selected-reaction-monitoring experiments. Parent ions of TCDD, TBDD, [13C1z]-2,3,7,8-TCDD, and [13C1z]-2,3,7,8-TBDDwere transmitted into the collision region by using MS1 in the selected-ion-monitoringmode at a resolution of about 500 (flat-top peaks). The resolution of Qzwas adjusted for baseline resolution. The resolution of Q2 is critical for comparing data since other investigators report resolution at full-width and one-half the height of the mass peak. On our instrument this results in approximately a 3-fold increase in sensitivity and can result in the detection of ions fl amu from the product ions of interest. In experiments to determine optimum conditions, using software supplied by the manufacturer, selected-reaction monitoring of the (M - COCl)+and (M - COB$ product ions was performed by synchronously stepping the voltages on MS2. Collision energies were varied between 10 and 50 eV in 10-eV steps or between 2 and 10 eV in 2-eV steps with sampling times of 10 ms followed
by a settling period of 10 ms. Pressures of nitrogen, argon, and xenon were varied in a random manner between 5 X lo" (no gas), 3 x IO", 1 x IO4, and 3 x lo4 mbar, as read 3 X lo4, 1 x on the ion gauge. Helium pressures were randomly varied between 5X 3 X lo4, and 3 x mbar. Readings on the Baynard-Alpert gauge, located outside the collision cell, are calibrated relative to nitrogen, requiring use of a correction factor to calculate true pressures of other gases. The corrected values are plotted in presentation of the data, and they must be further multiplied by 80 for helium, 250 for argon, 210 for nitrogen, and 450 for xenon. These are constants that account for the effective pumping speed (216.5 L/s) and molecular conductance of the gas. The total molecular conductance is the sun of the conductance through the entrance and exit apertures and is calculated from the following equation (10): C = 3.6A(T/M)1/2/1 + (3/16)(Hl/A) where, A is the cross sectional area of the aperture, in this case r = 0.105 cm for the exit aperture and r = 0.150 cm for the entrance aperture, T i s the temperature in the collision cell (K), assumed to be 293 K, M is the molecular weight of the gas, H is the perimeter of the entrance and exit apertures, and 1 is the length of the canal (plate thickness of the aperture). A set of standard conditions were chosen to determine the reproducibility of the measurements, and experiments using a particular gas were completed in one day. Estimate of Experimental Variation. The response of 1,3,6,8-TCDD was monitored on the first mass analyzer (MS1) to determine variations in MS1 response that might arise from instrument instability or injection errors. Any additional measurement errors, due to changes in detection or transmission in MS2 were calculated as the difference between the percent relative standard deviation (RSD) from the response measured on MS1 and the RSD measured for the m / z 259 and 393 product ions. The variations in the response of MS2 was used to estimate the error on MS1 and MS2. An overall estimate of reproducibility was calculated by summing the RSDs of measurements taken under the same pressure but under different collision energies and calculating the mean RSD. Finally, this mean RSD for each pressure was summed for all pressures to yield a mean RSD for the entire experiment. Full-Scan Collision-Induced Dissociation Experiments. The compounds were introduced via the direct-insertion probe with helium inlet into the ion source via the gas chromatograph to maintain conditions equivalent to GC/MS introduction. Collision-induced dissociation (CID) full scans were performed by setting the magnetic field to transmit the M + 2 ion of 2,3,7,8-TCDD ( m / z 322) or the M + 4 ion of TBDD ( m / z 500) to the collision region. The second mass analyzer was scanned over a mass range of 60-550 amu at a scan speed of 20 s for a given collision energy. Collision energies were varied either from 5 to 100 eV in 5-eV steps or from 2 to 100 eV in 2-eV steps for each mass scan. Resulting data are plotted as the intensity of a fragment normalized to the maxima of the m / z 259 (TCDD) or m / z 393 (TBDD) product ion to correct for changes in the rate of sample flow into the ion source. MS/MS Sensitivity Experiments. The M and M + 2 ions of native and isotopically labeled TCDD and the M + 2 and M + 4 ions of native and isotopically labeled TBDD, were monitored in determining the detection sensitivities under conditions previously found optimal for the formation of the (M - COCl)' or (M - COBr)+ product ions of TCDD and TBDD, respectively. The detection sensitivity was calculated by dividing the signal height by the height of the peak-to-peak baseline noise.
RESULTS AND DISCUSSION Precision of Measurements on MS1 and MS2. The RSD from the mean area of 1,3,6,8-TCDD on the first mass analyzer ranged from 7 t o 13% in all the experiments for 50-100 pg of TCDD injected. This demonstrates variations in the response which are in all likelihood due to injection variation. In experiments that used argon and helium as collision gases, eleven and six replicate measurements were made at the reference pressure and duplicate measurements made at all other pressures. The overall mean relative
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
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Collision Energy (eV) oNo gas + a 1 0 6 07x106
~2x105 ~7x105 02x10~
Flgure 1. Effect of collision energy and argon pressure on the formation of the (M - COCI)' and (M - COBr)' product ions of TCDD and TBDD. (Pressures of argon in the collision cell are approximately 250 times the values shown).
standard deviation of the response (area) that represents the total error of the measurements, was 11% for TCDD and 16% for TBDD (Ar) and 17% for TCDD and 25% for TBDD (He). The difference between these values and the relative standard deviation from the mean response of 1,3,6,8-TCDD is small (less than 18%)demonstrating comparable instrument stability for both mass analyzers. Due to the excellent precision among these measurements, the number of replicate measurements taken under the standard conditions was reduced to five in the experiments that used nitrogen and was reduced to three in the experiments that used xenon. The overall relative standard deviation from the mean response was 17% for TCDD and 25% for TBDD (N,) and 23% for TCDD and 51% for TBDD (Xe). The consistently larger RSD from the mean response for the (M - COBr)+ product ion was due to weaker signals (lower S:N). Once the optimal collision energy is established, the collision energy will not be varied across the mass peak. Thus the precision of these measurements can improve under static collision energy conditions since more time is spent sampling the signal, thereby increasing the signal to noise ratio. Optimum Collision Energy and Collision Gas Pressure Conditions. The results of selected-reaction-monitoring experiments of the (M - COCl)+ and (M - COBr)+ product ions were used to determine the optimum collision energy and collision gas pressure conditions in the presence of helium, argon, nitrogen, or xenon. In general, below about 30 eV the response of the m / z 259 and 393 ions increases as the pressures of argon or nitrogen are raised in the collision cell. Furthermore, the highest response shifts to lower collision energies as the pressure is increased (Figures 1 and 2). For example,
I
I
20
I
I
1
40
Collision Energy (eV) oNo gas +3x106 01x10-5 ~ 3 x 1 0 5 xlxlO-4 03x104 Figure 2. Effect of collision energy and nitrogen pressure on the formation of the (M - COCI)' and (M - COBr)' product ions of TCDD and TBDD. (Pressures of nitrogen in the collision cell are approximately 210 times the values shown.)
a shift from a collision energy optimum of 40-20 eV for the (M - COCl)' ion is observed as the argon pressure is increased from a pressure of 2 x lo4 to 7 X lo6 mbar. A different trend is noticed for the response of the (M - COBr)+ ion of TBDD. In this case, the collision energy maximum appears to be greater than 50 eV at pressures lower than 7 X mbar. In the presence of nitrogen, broad collision energy maxima are evident under the mid-pressures for the (M - COCl)+ product ion. The presence of broad collision energy maxima can be desirable in conducting quantitative analyses, since small changes in the collision energy due to instrument instability will not cause large changes in response. A compromise between response and instrument stability may therefore be necessary in determining optimum conditions. In the presence of helium or xenon, the response rapidly decreases with increasing collision energy (Figures 3 and 4). These experiments were repeated by varying the collision energies from 2 to 10 eV at helium and xenon pressures that provided the greatest response in the higher collision energy experiments. The optimum conditions found for TCDD were a xenon pressure of 1 X mbar and collision energy of 10 eV and a helium pressure of 2 X lo4 mbar and collision energy of 8 eV. A xenon pressure of 4 X lo4 mbar and collision mbar and energy a t 6 eV and a helium pressure of 2 X collision energy of 10 eV were found optimum for formation of the (M - COBr)+ product ion (data not shown). A summary of the optimum collision energy and collision pressure are presented in Table I. Optimum conditions, as shown in Table I, are those conditions (collision energy and collision gas pressure) that produce the maximum response. The center of mass collision energies calculated for these conditions are also shown, and it is gratifying that the values
716
ANALYTICAL CHEMISTRY, VOL. 63,NO. 7, APRIL 1, 1991 100-
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n N o gas ~ 1 x 1 0 6 04x106 n i x 1 0 5 ~ 4 x 1 0 5 71x104 Flgure 3. Effect on collision energy and xenon pressure on the formation of the (M COCI)' and (M COBr)' product ions of TCW and TBDD. (Pressures of xenon in the collision cell are approximately 450
-
times the values shown.) Table I. Summary of Collision Energy and Collision Gas Pressure Optima
collision gas helium nitrogen argon xenon
collision collision pressure, energy, eV mbar E,,, eV TCDD TBDD TCDD TBDD TCDD TBDD 8 30 20
10
io 20 20 6
2 x 10-5 2 x 10-5 1 x 10-4 3 x 10-4
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Collision Energy (eV)
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0.10 2.40 2.21 2.89
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~
0.08 1.06 1.48 1.25 ~
for nitrogen, argon, and xenon are similar to each other. Due to the low mass of helium, a collision energy of about 200 eV is needed to provide a similar center of mass collision energy. Subsequent experiments were conducted to determine the attenuation of ion beams of PFK, TCDD, and TBDD, so that optimum pressure conditions can be related to ion beam attenuation values that are independent of instrument type. A 50% attenuation of the m / z 331 ion beam of PFK was achieved at an indicated pressure of 7 X mbar for argon, 1X mbar for nitrogen, and 4 X mbar for xenon. A main-beam attenuation of 70% was obtained under a helium pressure of 2 X mbar. Under optimum pressures for formation of the (M - COCl)+ product ion of TCDD shown in Table I, main-beam attenuation of the parent ion of 50% was obtained in the presence of argon or nitrogen and 90% when xenon or helium was used as collision gases. These values were somewhat different for TBDD. A 20% attenuation was obtained for the m / z 500 parent ion of TBDD in the presence of argon or nitrogen and 95% main-beam attenuation when xenon was used. No attenuation of the parent
oNo gas + 2 x l 0 5 06x105 ~ 2 x 1 0 4 Figure 4. Effect of collision energy and helium pressure on the formation of the (M - COCI)' and (M - COBr)' product ions of TCW and TBDD. (Pressures of helium in the collision cell are approximately 80 times the values shown.) ion beam was observed under helium pressures of 2 x 10" mbar. This exercise demonstrates that optimum pressure conditions obtained by a systematic approach can be different from the pressure obtained by attenuation of a PFK ion beam to 50%. Effect of Collision Energy, Collision Gas Pressure,and Nature of the Collision Gas on the Formation of the Product Ions of TCDD and TBDD. The previous experiments were designed to determine the optimum conditions for selected-reaction monotoring of the (M - COCl)' and (M - COBr)+ product ions of TCDD under realistic conditions. The disadvantage of these experiments, however, was that observed trends could not be explained solely on observations based on the response of the product ions of interest. Thus, in order to understand the effect of collision energy, collision gas pressure, and the nature of the collision gas on formation of product ions, experiments were conducted by acquiring parent-product scans from 5- to 100-eV collision energies at the pressure investigated in the previous experiments. For purposes of clarity, selected data are presented a t three representative pressures and data not shown but pertinent to trends in product ion formation may be included in the discussion. CID of TCDD. Under unimolecular dissociation conditions, two product ions at m / z 287 [(M - Cl)+] (major fragment) and m / z 259 [(M - COCl)+]were observed. An overall decrease in dissociation occurred at the collision energy was increased, without changing the relative ratios of the two products. In the presence of gas, another major product ion at m/z 196 [(M - 2COC1)+]was formed. Under high collision energy (>50 eV) and high collision gas pressures (indicated pressure >1 X lo4 mbar), collision-induced dissociation of TCDD using argon,
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
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Figure 5. Effect of collision energy and argon pressure on the formatbn of the TCDD product ions. (Intensity on the y axis is normalied to the maxima of m l r 259.)
Figure 8. Effect of collision energy and nitrogen pressure on the formation of the TCDD product ions. (Intensity on the y axis is normalized to the maxima of mlz 259.)
nitrogen, and xenon produces other product ions a t m/z 231 [(M - COCl - COP], m/z 159 [(M - 2COC1- 2H)+], m/z 146 [(M - 2COC1- CCl - 4H)+],m / z 134 [(M - 2COC1- C1- 2C - 3H)+], m / z 123 [(M - 2COC1- 2C1- 3H)+], m / z 111 [(M - 2COC1- 2C1- C - 2H(+], m / z 99, and m / z 76. For the purpose of this discussion, we will consider trends in formation of the major product ions (Figures 5-8). In general, as the pressure of argon, nitrogen, or xenon is increased in the collision cell, greater production of the (M - COCl)+] ion occurs and the collision energy maximum and the collision energy that result in the (M - COCl)+ ion becoming the major product decrease. For the lowest pressures tested, higher pressures of nitrogen are required compared to argon for predominance of the (M - COCl)+ ion (Figures 5 and 6). The shift to lower collision energy optimum with increasing argon, nitrogen, or xenon pressure, observed in Figures 5-7, may be ascribed to greater production of the m/z 259 ion compared to the other product ions, as greater total energy is deposited in the parent through multiple collisions. The decrease in response a t collision energies >50 eV with increasing argon pressure is due to production of lower mass fragment ions, apparently as a consequence of "excess" energy being further utilized for multiple-bond breaking. The level response at low nitrogen pressures in Figure 6 can be explained by insufficient energy a t single collisions to cause fragmentation for production of the (M - COCl)+ product ion. When nitrogen is used, broader collision energy optima, compared to argon, occur because similar changes in the collision energy
correspond to smaller changes in the center of mass collision energy, thereby resulting in less fragmentation of the parent ion under equivalent conditions. Since xenon is a heavier gas, the collision energy optimum for production of the m / z 259 product ion is lower than for either argon or nitrogen. In contrast to the other gases, helium pressure had little effect on the degree to which the major product ions were formed (Figure 8). A decrease in the absolute response was observed that was not associated with greater production of low-mass product ions. This occurrence indicates t h a t high helium pressures may affect ion transmiasion and/or detection on our instrument or that the relatively light He may not impart sufficient internal energy into the parent ion to induce fragmentation. CZD of TBDD. Unimolecular dissociation of TBDD produces product ions corresponding to (M - Br)+ a t m/z 421, (M - COBr)+ at m/z 393, and (M - 2Br)+ at m / z 342. Under these conditions the m / z 393 product ion is the predominant ion. In the presence of gas, CID of TBDD produces other product ions observed a t m / z 314 [(M - COBr - Br)+], m / z 296 [(M - 2COBr)+], m / z 261 [(M - 2HBr - COBr)+], m / z 233 [(M - 2HBr - COBr)+], m / z 205 [(M - 2COBr - Br)+], m / z 182 [(M - 2HBr - Br)+], and m / z 124 [(M - 2COBr 2Br - 4H)+]. Trends in product ion formation using argon, nitrogen, xenon, or helium are shown in Figures 9-12 as plots of response versus collision energy. Similar to TCDD, the collision energy maximum for formation of the m / z 393 product ion
718
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991 I54
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decreases with increasing pressures of argon, nitrogen, and xenon in the collision cell. Changes in helium pressure have little effect on the collision energy optimum or the extent of fragmentation that cannot be explained by more extensive fragmentation to other progeny, again suggesting that high pressures of helium affect ion transmission and/or detection. Summary of CID of TCDD and TBDD. Certain general statements can be made that summarize trends observed in the CID of TCDD and TBDD. Increases in argon, nitrogen, or xenon pressures results in a decrease in the collision energy necessary for optimum production of the (M - COCl)+ and (M - COBr)+ product ions. At equivalent pressures, the optimum collision energy for formation of the (M - COCl)+ and (M - COBr)+ product ions follows the order of He Xe < Ar N2. The similarity in the optimum collision energy between argon and nitrogen suggests the importance of collision diameter (2.65 8, for helium, 2.94 A for argon, 3.15 8, for nitrogen, and 4.02 8, for xenon) (11)and mean free path in the CID of TCDD and TBDD. The mean free path is inversely proportional to the square of the collision diameter and thus a t the same pressure the number of collisions will be lowest for helium, about the same for nitrogen and argon, and highest for xenon. Fragmentation of TBDD is more facile than TCDD, presumably due to the weaker bond strength between carbon and bromine (276 kJ mol-') than carbon and chlorine (328 kJ mol-') (12). Detection Sensitivities for CID of 2,3,7,8-TCDDand 2,3,7,8-TBDD. Detection sensitivities, based on signahoise measurements were determined under the optimum collision
-
-
0
mlz 287
+
m/z 259
0
100
m/z 196
Flgure 8. Effect of collision energy and helium pressure on the formation of the TCDD product ions. (Intensity on the yaxis is normalized to the maxima of m l z 259.)
Table 11. Detection Sensitivities Measured on 1 pg of TCDD and 100 pg of TBDD compd
TCDD
gas
pressure, mbar
CE, eV
SN
He
2 x 10-5 1 x 10-4 I x 10-5 I x 10-5 2 x 10-5 2 x 10-4 I x 10-5 4 x 10-6
8 30 20 10 10
2:1 9:1 I:1 1O:l 12:l 7:1 12:l 11:l
NZ
AI Xe
TBDD
He
NZ
Ar Xe
I 20 6
energy and collision gas pressure conditions. The results are presented in Table 11. The detection sensitivities for the (M - COCl)+ product ion of TCDD range from 2:l to 1O:l for 1 pg and for the (M - COBr)+ product ion of TBDD range from 7:l to 12:l on 100 pg and are similar, under optimum collision energy and pressure conditions in the presence of nitrogen, argon, or xenon for both product ions. For helium the detection sensitivity is lower for the (M - COCl)' product ion but is similar for the (M - COBr)+ product ion compared to the other gases. This may be rationalized by differences in the He CID of TCDD and TBDD where fragmentation of TBDD was more facile than for TCDD. The higher detection sensitivities on the (M - COCl)+ ion of TCDD compared to the (M - COBr)+ ion of TBDD can be explained by more extensive fragmentation to other progeny and lower detection
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
719
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Flgure 9. Effect of collision energy and argon pressure on the formation of the TBDD product ions. (Intensity on the yaxis is "aiked to the maxima of m / r 393.)
Figure 10. Effect on collision energy and nitrogen pressure on the foination of the TBDD product ions-. (Intensity on the y axis is normalized to the maxima of m l r 393.)
sensitivities on MS1 for TBDD (a S:N of 1O:l for 2 pg of 2,3,7,&TBDD compared to 50:l for 1pg of 2,3,7,&TCDD. The detection sensitivities reported here are on the same order as a S:N of 1 0 1 for 500 fg of 2,3,7,8-TCDD reported by Reiner et al. (9), since it appears that these investigators calculated the S:N by dividing by half the peak-to-peak baseline noise. These values are about 5 times less than a S:N of 50:l for 1 pg determined by high-resolution gas chromatography/highresolution mass spectrometry (HRGC/HRMS). In another experiment, detection sensitivities were measured on an National Bureau of Standards (NBS) reference material by HRGC/HRMS (resolving power = 10000) and high-resolution gas chromatography/mass spectrometry/mass spectrometry (HRGC/MS/MS), using argon and nitrogen as collision gases. In these experiments, we obtained S:N values on 1 pg by extrapolation of measurements made on 34 pg of 2,3,7,8-TCDD. Signa1:noise ratios of 50:l were achieved by HRGC/HRMS. Signa1:noise ratios obtained by HRGC/ MS/MS were 6:l under an argon pressure of 7 X mbar and a collision energy of 20 eV, 8 1 under a nitrogen pressure of 1 X lo4 mbar and a collision energy of 30 eV, and 3 1 under a nitrogen pressure of 3 x mbar and a collision energy of 20 eV. In comparison, the manufacturers specification is a S:N of 1001 on 1 pg of 2,3,7,8-TCDD, and in a document ( I 3 ) ,the instrument company reported a S:N value of 79:l on 500 fg of 2,3,7,8-TCDD while monitoring the same ions. In our laboratory, the S:N measurements were made after extensive use of the instrument for fast-atom-bombardment analyses and minimal maintenance (e.g., analyzer bake-out and cleaning the ion source). We believe that the S:Nvalues
obtained on our instrument therefore represent those that can be achieved without extraordinary effort. A 2.5-fold decrease in sensitivity occurs while operating under nitrogen pressure and collision energy conditions that produce a broad collision energy maximum compared to conditions that result in a narrow collision energy maximum.
SUMMARY Experiments were conducted on a hybrid mass spectrometer to observe effects of collision energy, nature of collision gas, and pressure on the formation of product ions of TCDD and TBDD and to determine the optimum conditions for selected-reaction monitoring of the (M - COCl)+and (M - COB$ product ions. The optimum collision energy and collision pressure depend on the center of mass collision energy. The collision energy maximum, in turn, is dependent on the collision gas pressure. A decrease in the optimum collision energy occurs with increasing pressures of argon, nitrogen, or xenon. For xenon, due to its higher mass relative to the other gases, the optimum collision energy to achieve a given center of mass collision energy is lower compared to the other gases. A decrease in response was observed for these gases a t the highest pressure as a consequence of more extensive fragmentation to progeny other than (M - COCl)+ and (M COBr)+ and possible scattering effects. Helium pressure had little effect on production of product ions. The choice of optimum conditions has an arbitrary component. Quantitative analysis on a large number of samples may dictate that the analyst choose conditions that compromise sensitivity but maximize instrument stability, whereas
720
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991 IO A
1x ~ O mbari - ~
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-
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0 om/.?
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e
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Figure 11. Effect of collision energy and xenon pressure on the formation of the TBDD product ions. (Intensity on the y axis is normalized to the maxima of m l z 393.)
in the analysis of samples containing low levels of TCDD, the analyst may choose to use conditions that provide maximum instrument sensitivity but are less desirable in terms of instrument stability. We expected that similar optimum collision energy would be obtained on hybrid and triple-quadrupole instruments since hew-energy collisions take place in the collision cells of both these instruments. Reiner et al. (9) reported an optimal collision energy of 25 eV for the formation of the (M - COCl)+ product ion from TCDD on a triple-quadrupole mass spectrometer. An estimate of the actual pressure in the collision cell is obtained by multiplying the optima argon gas pressure by 250, providing a pressure of 1.8 x lo-* mbar on the hybrid mass spectrometer compared to an optimal pressure of 2.2 x IO9 mbar on a triple-quadrupole mass spectrometer. Under these conditions, the ratio of the parent ion to the (M - COCl)+ product ion is about 1:l on a triple-quadrupole mass spectrometer versus 18:l on a hybrid mass spectrometer. Further studies are needed to address the cause of these differences in order to standardize low collision energy MS/MS methods. Standardization of optimization procedures is needed for the development of generic MS/MS methods (8). Ideally, conditions should be used that best approximate "real" conditions of analyses. This approach is represented by experiments in this study conducted by HRGC/MS/MS by using selected-reaction monitoring. Unfortunately, these experiments do not provide data on the effect of collision energy,
2C 4C 6C 8C Collision Energy (eV)
.miz 393
+
m i z 342
IOC
miz 124
Figure 12. Effect on collision energy and helium pressure on the formation of the TBDD product ions. (Intensity on the y axis is normalized to the maxima of mlz 393.)
the nature of the collision gas, and the collision gas pressure on formation of product ions other than the specific ions monitored. Experiments that use the direct-insertion probe as a means to introduce pure compounds of the analyte, rather than, for example, PFK or PFTBA, are useful for obtaining this type of data (9). If this approach is taken, we have found that a small amount of helium let into the ion source from the gas chromatograph provides representative conditions for subsequent MRM analyses. Finally, because the collision energy optima are dependent on the collision gas pressure and collision pressures required to achieve a main beam attenuation of a PFK ion to 50% differ from the optimum, the approach used in our study provides more extensive information regarding optimal conditions for MS/MS analysis than approaches used in other laboratories.
ACKNOWLEDGMENT We thank L. Abbey, S. A. Guyan, and Y. Tondeur, Triangle Laboratories Inc., and B. N. Green, VG Analytical Inc., for helpful discussions; J. R. Hass, Triangle Laboratories, Inc., and R. Boyd, National Research Canada, for reviewing the manuscript; and Chris Porter, VG Analytical Inc., Mark Riley, VG Instruments, Inc., and John Riley, University of North Carolina, for their assistance in determining actual pressures in the collision cell and Northrop Services Inc. for providing us with standards of TCDD and TBDD. We also wish to thank the reviewers for their comments. LITERATURE CITED ( 1 ) Tondeur, Y.; Niederhut, W. E.: Campana, J. E.; Missler, S. R. Biomed. Environ Mass Spectrum. 1907, 14, 443-447. .I
Anal. Chem. 1991, 63, 721-725
(2) Charles, M. J.; Been, B.; Tondeur, Y.; Hass, J. R. Chemosphere 1989, 79 (I-6),51-57. (3) Charles, M. J.; Tondeur, Y. Environ. Sei. Technol. 1991, 24 (12),
1856-1860. (4) Busch. K. L.; Glish, G. L.; McCluckey, S. A. Mass SpectrometrylMass Spectrometry: Techniques and Applications of Tandem Mass Spec trometry; VCH Publishers, Inc.: New York, 1988; Chapter 3. (5) Dawson, P. H.; Douglas, D. J. Tandem Mass Spectrometry; McLafferty. F. W., Ed.; John Wiley & Sons: New York, 1983; Chapter 6. (6) Huang, L.; Tomer, K. 6.; McGown, S.; Moore, C. Presentation at the 10th International Conference on Organohalogen Compounds, Sept. 10-14. 1990, Bayreuth. Germany. (7) Scheiienberg. D. H.; Bobbie, 8. A.; Reiner, E. J.; Taguchi, V. Y. Proceedings of the 38th American Society of Mass Spectrometry (ASMS) Conference on Mass Spectrometry and Allied Topics; 1988. (8) Martinez, R . I . Rapid Commun. Mass Spectrum. 1988, 2 (I), 8-13.
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(9) Reiner, E. J.; Schelienberg, D. H.; Taguchi, V. Y.; McCurvin, D. M.; Clement, R. E. Proceedings of the 37th ASMA Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, 1989. (IO) Samson, J. A. R. Techniques of Vacuum Ultraviolet Spectroscopy; John Wiiey and Sons: New York, 1987. (11) Weast, R. C., Astle, M. J., Eds. CRC Handbook of Chemistry and Physics; CRC Press Inc.: Boca Raton. FL, 1982-1983. (12) Pauling, L. General Chemism; W. H. Freeman and Co.: San Francisco, 1970. (13) Analysis of polychlorinated dibenzo-pdioxins and dibenzofurans. VG Analytical Organic Mass Spectrometry; VG Analytical Limited: Wythenshawe. Manchester, M23 9LE England, 1987.
RECEIVED for review July 2,1990. Revised manuscript received November 21, 1990. Accepted December 17, 1990.
Quantitation Using Benzene in Gas Chromatography/Chemical Ionization Mass Spectrometry Charles Allgood,l Yee Chung Ma, and Burnaby Mumon*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
Dllute mixtures of benzene in helium provlde abundant C,H,'+ Ions, which selectlvely react by charge transfer under chemlcal ionlzatlon ( C I ) condltlons with unsaturated compounds in complex hydrocarbon mlxtures and do not react with alkanes or cycloalkanes. The charge-transfer spectra from the ion/molecule reactions of C,H~*+are very simple, containing essentially only M" Ions from the samples. Relative molar sensitivities for olefins and alkylbenzenes In benzene chemical ionizationmass spectrometry (CIMS) are essentlally constant within f10-15 %, Independent of molecular welght In the range of 100-200 Da, molecular structure, the degree of substitution, or Ionization energy. The relative molar sensitlvltles In benzene CIMS show much loss varlatlon with molecular structure than relative molar sensnlvltles In low-voltage electron ionization mass spectrometry (LV-EIMS). GCKIMS with benzene as the charge-transfer reagent gas allows quantitative analyses of the aromatlc and olefinic components In gasolines or other complex hydrocarbon mlxtures without the necesslty of calibration curves for the individual components.
INTRODUCTION Benzene has several advantages that make it an attractive reagent gas for the analysis of complex mixtures using gas chromatography/chemical ionization mass spectrometry (GC/CIMS). The ionization energy of benzene is 9.25 eV, about in the middle of the range of ionization energies for most organic compounds (1-3). Thus, benzene ions should undergo charge-transfer reactions with unsaturated hydrocarbons that have ionization energies lower than 9.25 eV. This selectivity for unsaturated compounds using benzene CI has been demonstrated previously (4-6). Since the proton affinity of the phenyl radical, 208 f 7 kcal/mol (3, 7) or 212 f 2 kcal/mol (3,8),is larger than the
* To w h o m correspondence should be addressed.
Present address: Organic Analytical Research Division, NIST, Gaithersburg, MD 20889.
proton affinities of most hydrocarbons ( 3 ) ,proton transfer from C&'+ to most hydrocarbons will be endothermic and consequently will not be observed. It has also been reported recently, for a small number of examples, that if both proton transfer and charge transfer from C6H6'+ions are exothermic, only charge transfer is observed (9). The appearance potentials of most fragment ions from alkylbenzenes and olefins are larger than 9.25 eV, the ionization potential of benzene (2); therefore, dissociative charge-transfer reactions of C6H6+ to give fragment ions are endothermic and should not occur. Consequently, benzene CI mass spectra of aromatic and olefinic hydrocarbons are predominantly one-peak spectra, containing essentially only M'+ ions. Quantitation requires sensitivity factors for each compound being analyzed: a tedious or impossible task for complex mixtures. It was noted previously that the relative rate constants for reactions of C6H6'+with several alkanes were essentially zero and that the rate constants for charge-transfer of C6H6'+with a few aromatic hydrocarbons (or the relative molar CI sensitivities of the compounds) were independent of the exothermicity of reaction, molecular structure, and molecular weight-essentially constant (10). It has also been reported that the molar sensitivities of aromatic hydrocarbons in chlorobenzene CI are independent of molecular weight and molecular structure (11). Consequently, one may use C6H6*+ as a CI reactant ion in GC/CIMS with little danger of mass interference from isobaric compounds (except for isomers) and with minimal need for calibration data. Low-voltage electron ionization mass spectrometry (LVEIMS) was developed many years ago for the quantitation of unsaturated hydrocarbons in complex mixtures (12-16). Early experiments showed that the relative sensitivities per gram for a homologous series (alkylbenzenes, for example) decreased with increasing length of the aliphatic side chain (13). Other experiments showed that the relative molar sensitivities for alkylbenzenes were independent of the length of the side chain and increased significantly with increasing number of alkyl substituents on the benzene ring (15). Consequently, in an analysis from a batch inlet system without separation of the individual compounds, the deter-
0003-2700/91/0363-0721$02.50/0 0 1991 American Chemical Society
