Instrumentation Ralph C. Dougherty Department of Chemistry Florida State University Tallahassee, Fla. 32306
Negative Chemical Ionization Mass Spectrometry Negative chemical ionization mass spectrometry (NCIMS) offers unique and interesting potential in analytical chemistry, studies of chemical reactiv ity, and in the development of the re lationship between experimental and theoretical chemistry. In the area of analytical chemistry, NCI has shown very high selectivity for particular classes of molecules and very high sen sitivity for those classes. It is possible by use of NCI to detect submicrogram quantities of specific compounds in matrices that contain microgram quantities of molecules of biological origin. Classes of molecules that have intrinsically high NCI sensitivities are molecules that are oxidizing agents,
alkylating agents, or both. These classes of molecules span a large por tion of the spectrum of the toxic sub stances that appear in the environ ment and, as a result, one of the major applications of NCIMS has been in the detection and analysis of toxic substances. In the area of chemical reactivity, NCI, along with other negative ion techniques, makes it possible to study the reactivity of anions in the absence of solvation. Inversions of anionic re activity ongoing from the gas phase to solution are by now very well known. Specialized techniques within the area of high pressure negative ion MS have made it possible in recent years to de
termine the electron affinities of a large number of molecules. Electron affinities give data about the energies of low-lying vacant orbitals, just as ionization potentials give information concerning the energies of the highest energy electrons in molecules. The op portunities for testing quantum me chanical theories of molecular struc ture by combining these techniques are only now being realized. In contrast to positive ion MS, neg ative ion MS has, until the last dec ade, been a poor and neglected shirttail relative. The reasons for the ne glect of negative ion MS, a technique with virtually the same potential for analytical and chemical reactivity
(b)
(a)
[A- + Β'
Interatomic Distance
Interatomic Distance »
Interatomic Distance Cl-C
Interatomic Distance C-CI
Figure 1. Negative ion-forming processes (a) Resonance capture of an electron to give a vibrationally excited molecule ion. (b) Dissociative capture of an electron to give a fragment ion and a neutral, (c) Ion-molecule association to give an anion molecule adduct. Lowest vibrational levels are in blue. Vertical dashed lines in (a) and (b) are Franck-Condon electronic transitions
0003-2700/81/0351-625AS01.00/0 © 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981 · 625 A
Table I. Ion-forming Reactions in NCIMS. (a) Reactions common to all systems. Ν
M + e s " — [M]—·-*• M-· AB + e s ~ — A ~ + B" (b) Hydrocarbon ( 9 3 % ) , methylene chloride or methylchloride ( 5 % ) , oxygen ( 2 % ) . Chloride/oxygen reagent. RCI + ZH + RCI + ZH + Ad-·
es" Cr CI" Cl~ + 02
— R• + C l " - * [ZH Cl]— RCI2_ —• 2T + HCI — A O _ + CIO"
This gas mixture has the same high sensitivity for electron-capturing molecules as pure hydrocarbon or hydrocarbon/oxygen mixtures, and it also has high sensitivity for alkylating agents such as aliphatic polyhalides, phosphate esters, and carbamates. The selectivity of the gas mixture is very high—neutral lipids are virtually transparent.
(c) Hydrocarbon ( 9 5 % ) , water ( 5 % ) . Hydroxide reagent. H 2 0 + e-T AH + O H M + OH-
O H - + HA - + H20 MOH~
— — -*
Hydroxide can also be generated by the following reactions: N20 + e s _ O " · 4- RH
N2 + O - · O H - + R-
— —
Reactions of hydroxide are mimicked by methoxide and amide reagents: CH 3 ONO + e s ~ NH 3 + e s -
—" -*
CH3O- + NO N H 2 - + H-
All these reagents give high sensitivity for many structural classes of compounds. The selectivity for toxic substances or electron-capturing molecules is low, because these reagents all react with neutral lipid esters and alcohols and can even produce intense spectra with saturated hydrocarbons.
(d) Fluorocarbon, CHF 3 , C 2 F 3 CI 3 , etc. Fluoride or chloride reagent. CHF 3 + e s ~ AH + F M + F" M - - + F-
— —· -* —
F - + 2F- + CH A " + HF MFM + F-
Fluorocarbons will produce either F~ or C I - as the reactive reagent, depending on their structure. CHF 3 gives F~, CF 2 CI 2 and CFCI 2 CF 2 CI give C I " with some C l 2 - · . Fluoride is an exceptionally strong gas phase base, and mimics O H " in reactivity and low selectivity. All of the fluorocarbon reagents appear to have decreased sensitivity for electron-capturing substances because of reactions like these: Μ-· + Ρ Cl2-· M - · + CI"
-* — —
z - + ci·
— z- + c r
M + F0 Γ + CI' Μ + 0Γ
Similar reactions can occur with other radicals with high electron affinities in the source.
studies as its positive ion counterpart, stem from the chemistry and physics of negative ion formation and detec tion. Problems associated with these two areas were only significantly over come within the last decade. Negative Ion Formation The fundamental difference be tween the ion-forming processes in
negative and positive ion MS is based on the fact that positively charged ions can retain relatively large amounts of excess internal energy for a significantly longer time than their negative ion counterparts. Other than abstraction of an electron from a near by neutral, a positive ion can do noth ing to neutralize its charge once it has been formed. Negative ions, on the
other hand, can merely eject an elec tron, and they do so readily if their in ternal energy exceeds the electron af finity of the neutral. NCIMS had its origins in studies of pressure enhancement of negative ion mass spectra using conventional mass spectrometers (1-3). These studies, together with earlier investigations of the energetics of ion formation in neg ative ion MS, provided a basis for un derstanding the fundamental ionforming processes that occur under the conditions of NCI—that is, nega tive ion MS in which the ion source is maintained at a pressure of approxi mately 1 torr. There are three impor tant ion-forming reactions that occur in NCIMS. These are: resonance cap ture of a thermalized electron (Figure la), dissociative capture of a low ener gy electron (lb), and ion-molecule re actions that occur between ions in the ion source and neutrals. A large num ber of specific ion-molecule reactions are known in NCI, including charge transfer, proton transfer, hydride transfer, oxygen exchange reactions with either halogens or hydrogen, and anion-molecule adduct formation. Anion-molecule adduct association is illustrated in Figure lc for the case of chloride attachment to methylene chloride. The common ion-forming re actions in NCIMS are summarized in Table I and discussed in the following paragraphs. The formation of ions by resonance capture of an electron is strongly de pendent upon the electron affinity of the substrate, the energy spectrum of the electrons producing the ionization, and the frequency of collision of mole cule ions and excited molecule ions with neutrals. The energy spectrum of the electrons in the ion source de pends on the energy of the primary ion beam, the nature of the gases in the ion source, the pressure, and the electric fields in the source. If all of these factors are carefully monitored and controlled, it is possible to obtain a reproducible electron energy spec trum in the ion source, and molecule ion sensitivities for a given compound can thus be reproduced from experi ment to experiment. Collisional relax ation of vibrationally excited states of newly formed molecule anions is di rectly dependent upon both the con centration of the anions in the ion source and the concentration of neu trals (reagent gas pressure). Once again, control of the pressure in the ion source is a critical parameter for obtaining reproducible sensitivities for molecule anions. In my laboratory, gas pressure is controlled by use of a com mercially available capacitance monometer that is isolated from ground and maintained at the same potential as the ion source. This monometer ac-
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981 · 629 A
400
120
140
160
-ι 180
1—r 220 200
240
260
280
300
320
340
360
380
400
420
m/e
Figure 2. Comparison of positive and negative CI spectra (a) Positive chemical ionization mass spectrum of a chicken extract containing Dieidrin. (b) NCI mass spectrum of the same extract under the same conditions. Reagent gases were isobutane and methylene chloride
curately monitors source pressures to 0.001 torr. Pressure is adjusted by use of metering valves on the gas feed lines, and it remains constant over the period of a working day. The efficiency of dissociative cap ture ion-forming reactions is strongly dependent upon the energy spectrum of the electrons in the ion source, be cause these reactions are generally sharply peaked with electron energy. For example, the optimum electron energy for formation of CH 2 ~- from methane is approximately 10.1 eV (4). The precise energy maximum for a given dissociative capture reaction de pends upon the energy difference be tween the virtual vacant orbitals of a molecule, the potential energy of the molecule itself, and the position of the intersection of the potential energy surfaces for the molecule and the dis sociating pair (see Figure lb). The vir tual vacant orbitals of a molecule are those orbitals that would be singly oc cupied if the bond in question was subjected to homolytic cleavage. The optimum energies for dissociative cap ture of an electron rarely exceed 12 eV. This is fortunate, as the num ber of low energy electrons drops steeply in the energy range between approximately 12 eV and the energy of the primary ionizing beam, usually 500 eV. The types and abundance of ionmolecule reactions that occur in NCIMS are strongly dependent on the concentrations and structures of the reagent gases employed (see Table I). Hydrocarbons and the noble gases
typically produce NCI mass spectra that are dominated by resonance cap ture and dissociative capture of elec trons by the analyte in the ion source. Gases that produce abundant quan tities of gas phase nucleophiles, such as methylene chloride (5), produce spectra that contain nucleophile adducts of the analyte in addition to ions that may be formed by electron cap ture processes. If reagent mixtures are employed that produce abundant quantities of strong gas phase bases such as hydroxide, methoxide, or fluo ride, the resulting NCI mass spectra generally reflect the result of acidbase chemistry in addition to electron capture processes and anion-adduct formation. Adduct formation occurs with hydroxide; however, the most common process is deprotonation. Hydroxide ion can be produced as a reactant by a number of methods in addition to dissociative electron cap ture by water (see Table I). In a re agent gas mixture containing nitrous oxide and methane (1:1), the reaction sequence that generates hydroxide in volves dissociative capture by nitrous oxide to give the oxide anion, which then abstracts a hydrogen atom from methane or any other abundant hy drogen species to give O H - . Using hy droxide ion as a reagent, NCI mass spectra can be produced from hydro carbons (6), ethers, alcohols, and neu tral lipids (7), all of which are normal ly transparent to NCI mass spectra taken with hydrocarbon or chloridecontaining reagent gases (5, 9). Methoxide ion, a weaker gas phase
630 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
base than hydroxide, can be generated as an NCI reagent by use of mixtures of hydrocarbon gases and methylnitrite (8). The analytical potential of this reagent has yet to be throughly investigated. Chlorofluorocarbons have been sug gested as a source of gas phase nucleo philes for NCIMS. In particular, dichlorodifluoromethane (Freon 12) and l,l,2-trichloro-l,2,2-trifluoroethane (Freon 113) have been suggested for this purpose. It has been our experi ence that NCI sensitivities for toxic substances or electron-capturing mol ecules have been much lower using these gases than when methylene chloride was the source of Cl~. The reason for this lower sensitivity is dis cussed in Table I, part d. It happens that fluoride radicals, chloride radi cals, or CF2CI" radicals all have higher electron affinities than all but a few molecules or radicals. The ensuing electron transfer reactions in the plas ma result in an increase in F~ or Cl~ at the expense of ions that would pro vide analytical information about the electron-capturing substances in the plasma. The remarkable selectivity of NCIMS using hydrocarbon or hydrocarbon/methylene chloride reagent gases is illustrated by the fact that methylstearate caused no change in the spectrum of a reagent gas of 0.45 torr isobutane and 0.05 torr methylene chloride when it was introduced into the ion source in 0.1-mg quantities. In contrast, compounds like DDT will produce identifiable chloride attach-
ment NCI mass spectra when introduced into the ion source in quantities as low as 1 ng. By use of multiple ion monitoring techniques, the detection limits for compounds like DDT can be pushed to sub-picogram levels. The selectivity cited above is illustrated by the chemical ionization mass spectra in Figure 2. These spectra were generated under identical ion source conditions from an extract of chicken tissue that contained approximately 0.1 ppm Dieldrin. The positive CI mass spectrum (2a) shows a homologous series of ions corresponding to neutral lipids that came through the Fluorosil cleanup procedure. There are also ions corresponding to Dieldrin minus chloride and protonated Dieldrin; however, their prominence in the spectra is limited. The NCI spectrum of this same extract obtained under identical ion source conditions is shown in Figure 2b. In this spectrum the only ion of any prominence corresponds to the chloride adduct of Dieldrin. The compound is easily identified by its molecular weight, negative mass defect, and chlorine isotope cluster. The selectivity of NCIMS stems from the fact that not all molecules have low-lying vacant orbitals or virtual vacant orbitals, in contrast to positive ion mass spectra, which are generated by removing valence level electrons, a feature common to all chemicals. The selectivity of NCIMS is rather strongly dependent upon the construction of the ion source and mass analyzer, and the reagent gas mixture. Paraffinic petroleum hydrocarbons are virtually transparent in NCIMS using hydrocarbon/methylene chloride reagent gas mixtures in a double-focusing mass spectrometer. These same hydrocarbons can, however, produce intense NCI mass spectra if hydroxide is used as the reagent to give hydroxide attachment ions (6). NCIMS Analysis
Within the last decade, two international conferences have been held on NCIMS. The proceedings of the most recent conference appeared very recently (10). It is worthy of note that virtually all of the analytical applications of NCIMS presented at that conference involved analysis of environmental samples. The reason for the extensive application of NCIMS to environmental chemistry stems from the selectivity of the technique when it is applied using hydrocarbon/methylene chloride- or oxygen-containing reagent gases. NCIMS has been successfully used for the analysis of trace metals in the atmosphere (11), the analysis of drugs and drug metabolites in biological samples (12), and the analysis of polychlorodibenzodioxins and related
Cle and Chlordane mlz 441 Nonachlor mlz 475
m#h-r 350 Mass Number Figure 3. NCI mass spectrum of an extract of 50 mg of Lake Ontario trout Reagent gases were isobutane and methylene chloride. Only negative mass defect ions are shown
compounds in biological samples (13), in addition to many other applications. The following paragraphs will focus on applications of NCIMS in screening environmental substrates for the presence of unknown toxic substances, especially polychlorinated organic compounds. The first step in residue screening is the clean-up of the samples. The paradigms that we have used for the development of screening procedures for toxic substances include the following: The procedure should be exceptionally simple, and solvent reduction steps should be limited, if possible, to one. Following this stricture will improve both reproducibility and recovery of low levels of toxic substances from the matrix. In screening procedures, the sample clean-up should avoid adsorption chromatography if possible. There are a number of polar toxic substances in environmental mixtures that simply will not survive adsorption chromatography when the component is present at residue levels. In the past we have avoided all use of gas chromatography for residue screening. Avoidance of gas chromatography has the advantage that direct probe NCIMS of the complex mixture that is obtained after sample clean-up can potentially detect unknown materials that would not survive gas chromatographic analysis, in addition to those materials that would require derivatization prior to gas chromatography. Avoidance of gas chromatography as a sample introduction technique for NCIMS has the disadvantage that concentration estimates for individual components taken from direct probe NCI mass spectra are considerably less reliable than those that would be obtained by GC-NCIMS techniques. The reason for this.difference in reliability in quantitation stems from the very substantial influence of the sample matrix
632 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 4 , APRIL 1981
on both the relative and absolute sensitivities for individual components in direct probe introduction of complex mixtures. For the moment, this dilemma more or less requires that different experiments be performed for general screening for thepresence of unknown compounds and for quantitation of compounds that are known to appear in the matrix. In the attempt to identify unsuspected toxic substances in environmental substrates, the screening design outlined above with direct probe NCI analysis has been a success. It is widely known that freshwater fish from highly industrialized areas have substantially higher concentrations of many organic toxic substances than their marine counterparts. Figure 3 illustrates the direct probe NCI mass spectrum, obtained with isobutane containing approximately 5% methylene chloride and 2% oxygen, of an extract of Lake Ontario trout that had been fractionated by gel permeation chromatography (14). This mass spectrum was obtained from an extract of 50 mg of tissue. Concentration levels in the matrix were in the part per billion range, indicating nanogram level sensitivity using scanning MS for the components illustrated in the figure. The presence of octachlorostyrene and heptachlorostyrene was not anticipated in this sample. These compounds were revealed by their oxygen exchange ions with appropriate negative mass defects and isotope ratios, at m/e 357 and 323. To our knowledge neither octachlorostyrene nor heptachlorostyrene are commercial products. The most probable source for these compounds in the environment is the production of chlorine gas using carbon anodes. Planar polychlorinated aromatic molecules often exhibit exceptional toxicity to higher organisms. The toxicities of 2,3,7,8-tetrachlorodibenzo-
500
100
i: I
CU m/z279
r^Oj^
Cb u mlz 301
/
o-
40
mlz 471
oc 2 0 -
250
500
Mass Number Figure 4. NCI mass spectrum of planar polychlorinated aromatic hydrocarbons isolated from Tittabawassee River (Michigan) carp
Were
the One···
CATALOG you need for Gas Chromatography. Our new 23-GC Catalog contains the latest products in four easy-to-use sections: • Gas Chromatography Supplies • Gas Chromatography Reagents • Biochemicals and Standards • Chemical Adsorbents Also look for our 23-LC Catalog soon!
If you are already receiving the APPLIED SCIENCE Newsletter, your catalog will be mailed automatically. Call if you are not on our mailing list. APPLIED SCIENCE is now consolidated in one location to give you the finest quality, delivery, and technical service available.
APPLIED SCIENCE Applied Science Division Milton Roy Company P.O. Box 440, State College, PA 16801 Phone: 814-466-6202
Reagent gases were isobutane, methylene chloride, and oxygen. Only negative mass defect ions are shown. C-13 isotopes are eliminated for low intensity clusters
p-dioxin (TCDD) and that of the related tetrachlorodibenzofuran are well known. To screen for the presence of the planar polychlorinated aromatics using direct probe NCIMS, it is necessary to eliminate the nonplanar polychlorinated organics from the matrix. This requirement is imposed because the nonplanar polychlorinated organics in environmental samples, for example, those detected in the spectrum in Figure 3, exist in environmental samples at concentrations up to three orders of magnitude higher than the planar molecules. The total ionization in NCIMS is limited by space charge. That is, there is an absolute maximum to the number of ions that can exit the ion source for any set of ion source conditions. Therefore, if the mixture to be analyzed contains abundant quantities of compounds that produce intense negative ion mass spectra, these are the only compounds that will be seen. By removing the abundant components from the mixture, it is possible to detect orders of magnitude lower quantities of other components that are also present. We isolated these compounds by adsorption on activated carbon. The carbon was then washed with a solvent sequence to remove nonplanar polychlorinated organics and neutral lipids, and the planar polychlorinated organics were desorbed using toluene as a solvent. Figure 4 illustrates the NCI screening mass spectrum of an extract of carp that was obtained from the Tittabawassee River in Michigan adjacent to the Dow Chemical manufacturing facility. This spectrum indicates the presence of polychlornaphthalenes as oxygen exchange ions and a series of polychlordibenzodioxins (15). The mass spectrum in Figure 4 does not
CIRCLE 8 ON READER SERVICE CARD 634 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
accurately reflect the relative abundance of the specific dioxins in the mixture. The sensitivity of NCIMS for TCDD is lower than for any of the other tetrachlorodioxin isomers, and it is also lower than the sensitivity of NCIMS for the higher chlorinated dioxins. The confirmation that the tetrachlorodioxin that appears at m/e 301 in the spectrum in Figure 4 is in fact the 2,3,7,8 isomer has been obtained by capillary gas chromatography, with both positive and negative ion detection (15,16). In a survey of fish samples taken from freshwater sources in the Midwest and Northeast for planar polychlorinated organics, we were able to detect polychlorinated naphthalenes (Halowax) in every fish sample. Polychlorinated dibenzofurans were detected in extracts of fish that contained concentrations of polychlorobiphenyls exceeding 2.5 ppm. The polychlorinated dibenzodioxins illustrated in Figure 4 were detected only in samples of fish taken from the Tittabawassee River. It seems highly unlikely that the trace chemistry of ordinary combustion is responsible for this dioxin contamination (17). We have been interested in screening human seminal plasma for the presence of toxic substances because of observations in the literature that suggest that male fertility potential in the U.S. has decreased during the past 30 years (15,18,19). Figure 5 illustrates the 12C negative mass defect ions that appeared in the NCI mass spectrum of an extract of human seminal plasma that was obtained using steam distillation with continuous liquid-liquid extraction as the clean-up procedure (14). This spectrum showed evidence of the presence of polychlorobenzenes, polychlorophenols, poly-
ο100
ο-
Χ
