Anal. Chem. 1992, 64, 1586-1593
1586
Electrochemical Origin of Radical Cations Observed in Electrospray Ionization Mass Spectra Gary J. Van Berkel,’ Scott A. McLuckey, and Gary L. Glish Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 -6365
A variety of experlmental data Is presented that Implicates electrochemkaloxidation of anaiytesInthe electrospray(ES) needle as the mechanismfor formation of molecular radkal catlons observed In the ES lonlratlon mass spectra of alkylsubstituted metalkporphyrlns, polycyclic aromatlc hydrocarbons (PAH’s), and other compoundtypes. Anaiyte structural characteristics and soiutlon-phase haw-wave oxidation p tentlab (whkh correlate with gas-phase lonlzatlon energies) can be usedto evaluatethe IlkeHhoodof forming and obrrenlng a particular compound as a radkal cation. Use of an appropriate solvent Is critlcal In the observation of radical catlonr genefatedby the ES process. I n addnlon to dkrokhg the anaiyte and providing a stable electrospray, the solvent( 8 ) must “stablllze” or otherwise protect the radkal catlon from reaction8 In solution. Appropriatesolvent systems (e& methylene chlorkle/O.l% trltluoroacetlc acid) are much the same as used In traditional studies of electrochemkally generated radkal cations. The ability to produce radkal cations In the ES process expands the utility of ES lonlzatlon mass spectrometryto Include compoundclasses not normally amenableto the technique (e.g., neutral, nonpolar compounds such as PAH’r) and providesfor generationof a different type of molecular species than normally produced In positlve-lon ES lonlratkn (Le., Me+versus (M H)’, (M Na)+, etc.).
+
+
INTRODUCTION Electrospray ionization mass spectrometry (ESMS), since ita introduction in the early 1980s,lv2 has emerged a an important technique for the analysis of a wide variety of compound types ranging from very high molecular weight biopolymers to metal ions (for reviews see refs 3-71, Electrospray is not, however, a universal ionization technique. Although details of the ES ionization mechanism are at is~ue,3fJ-~~ empirically it has been found that the best analytical results are obtained for compounds that are ionic in solution (i.e., performed ions). Such species include, for example, metal salta,14organic ~ a l t a , ’ ~and 1 ~compounds (e.g., (1) Yameahita, M.; Fenn, J. B. J. Phys. Chem. 1984,88,4451. (2) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984,88,4471. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. K.; Whitehouse, C. M. Science 1989, 246, 64. (4) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. K. Mass Spectrom. Reu. 1990, 9, 37. (5) Smith, R. D.; Loo,J. A.;Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990,62,882. (6) Huang, E. C.; Wachs, T.; Conboy, J. J.; Henion, J. D. Anal. Chem. 1990,62, 713A. (7) Mann, M. Org. Mass Spectrom. 1990,25, 575. (8) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976,64, 2287. (9) Thomson, B. A.; Iribarne, J. V. J. Chem. Phys. 1979, 71, 4451. (10) Schmelzeisen-Redeker, G.; Btitfering, L.; R6llgen, F. W. Int. J. Mass Spectrom. Ion Processes 1989, 90, 139. (11) Smith, D. P. H. IEEE Trans. Ind. Appl. 1986, IA-22, 527. (12) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1991, 63, 1989. (13) Blades, A. T.; Ikonomou, M. G.; Kebarle, P. Anal. Chem. 1991, 63, 2109. (14) Jayaweera,P.; Blades, A. T.; Ikonomou, M. G.;Kebarle, P. J.Am. Chem. SOC.1990,112,2452.
peptides and proteins) incorporatingfunctionalities that can be ionized via solution-phase Bronsted or Lewis acidlbase chemistry (see, e.g., refs 3-7). The latter species are typically observed as protonated, sodiated, or otherwise cationized molecules in positive-ion mode and as the deprotonated molecules or as molecular adduct anions in negative-ion mode. Ionization of neutral, relatively nonpolar compounds, such as aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), by means of solution-phase acid/base chemistry, is inefficient. Therefore,these types of compounds either show reduced sensitivity or are not detected under normal ESMS conditions.18 Recently,we reported the observation of molecular radical cations in the ES mass spectra of various alkyl-substituted metall~porphyrins.’~This observation was significant because molecular ions, formed by loss or gain of an electron, i.e., radical cations and radical anions, are generally not observed in true ES ionization mass spectra &e., when no gas-phase ionization processes are at work). Tentatively, we interpreted our observations as an indication of either chemical oxidation of the porphyrins involving some species in the ES solvent system, prior to or during the spraying process, or electrochemical oxidation in the ES needle. Both explanations seemed plausible since chemica12+22 and electrochemica121-26 oxidation of metalloporphyrins and the stability of metalloporphyrin radical cations in solution*26 are well documented. These preliminary explanations for radical cation formation suggested that alternate types of solution chemistry, or a poorly understood phenomenon in the ES process, might be exploited to form ions in solution and successfully detect compounds not normally amenable to ESMS. This reaoning led, for example, to our use of an alternative chemical method, viz. charge-transfer complexation, to form ions in solution from compounds such as N,N,”,N’-tetramethy1-1,4-phenylenediamine, 2,3-benzanthracene, and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone for analysis by ESMS.18 In this paper, data are presented that implicate electrochemical oxidation at the metallsolution interface of the ES needle, or metal connector used as a high-voltage contact, as another mechanism for formation of the molecular radical cations in ES ionization. Furthermore, electrochemical ~~
(15) Bruins, A. P.; Weidolf, L. 0.G.; Henion, J. D.; Budde, W. L. Anal. Chem. 1987,59,2647. (16) Udseth, H. R.; Loo, J. A.;Smith, R. D. Anal. Chem. 1989,61,228. (17) Conboy, J. J.; Henion, J. D.; Martin, M. W.; Zweigenbaum, J. A. A d . Chem. 1990,62,800. (18) VanBerkel, G. J.; McLuckey, S. A.;Glish, G. L. Anal. Chem. 1991, 63, 2064.
(19) VanBerke1,G. J.; McLuckey,S.A.;Glish,G.L.Anal. Chem. 1991, 63, 1098. (20) Fuhrhop, J.-H.; Mauzerall, D. J. Am. Chem. SOC. 1968,90,3875. (21) Felton, R. H.; Dolphin, D.; Borg, D. C.;Fajer, J. J.Am. Chem. SOC. 1969, 91, 196. (22) Fuhrhop, J.-H.; Mauzerall, D. J.Am. Chem. SOC. 1969,91,4174. 1970, 92, 2982. (23) Wolberg, A.; Manassen, J. J. Am. Chem. SOC. (24) Fuhrhop, J.-H.; Kadish, K. M.; Davis, D. G. J.Am. Chem. SOC. 1973,95,5140. (25) Dolphin, D.; Muljiani, Z.; Rousseau, K.; Borg, D. C.; Fajer, J.; Felton, R. H. Ann. N.Y.Acad. Sci. 1973,206,177. (26) Smith, K. M., Ed. Porphyrins and Metalloporphyrins; Elsevier: Amsterdam, 1975.
0 1992 A m l c a n Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
. -
1587
!rture
solvent/ needle (5cm long) S.S.
syringe Pump metal connector
solvent/
syringe Pump connector
solvent/
syringe Pump
front aperture plate
Flgure 1. Schematic representations of the three different ES source geometries wed in these experiments. I n setup a the analyte solution is sprayed from a 120-pm4.d. (500-pm-o.d.) stainless steel needie at which point the high voltage necessary to produce a spray Is applied to the solution. I n setup b the high-voltage contact is made several centimeters up stream via an in-line metal connector and the solution is sprayed from the end of a 5-cm length of 100-pm4.d. (350-pm-0.d.) fused-silica capillary. In setup c the high-voltage contact is made several centimeters up stream via an in-line metal connector and the same needle used in setup a and the solution is sprayed from the end of either a 1 5 , 30-, or 60Gm length of 100-pm4.d. (350-pm-0.d.) fused-silica capillary. Drawings are not to scale.
formation of radical cations from organic molecules in the ES needle is shown to be consistent with the electrophoretic charge separation description of the ES process recently put forward by Kebarle and c o - ~ o r k e r s . ~ For ~ J ~ the electrophoretic charge separation mechanism to be operational, in the positive-ion mode, charge balance requires that an oxidation reaction, ultimately resulting in electron flow to ground, must occur at the metal/solution interface within the ES needle (or at the metal high-voltage connection to the solution) to balance the positive-ioncharge leaving the needle in the spray. The data in this paper demonstrate that for some types of analytes, under appropriate conditions, a major oxidation product can be the molecular radical cation of the analyte. Conditions necessary for the observation of radical cations for a number of different analytes and analytical applications of this ES ionization phenomenon are discussed.
EXPERIMENTAL SECTION
AU experiments were carried out with a Finnigan-MAT ion trap mass spectrometer (ITMS) modified for atmospheric sampling that has been described in detail.199n928The results and experiments described here are dependent on solution chemistry and the ES process and, therefore, should be largely independent of the mass spectrometer used in the analysis. In the normal ES mode of operation, a syringe pump (Harvard Apparatus, Inc., Cambridge, MA) was used to pump a solution of the analyte through a short length of 500-pm4.d. Teflon tubing at a rate of 1-5 pL min-l and then through a 5-cm-long dometipped 120-pm4.d. (500-pm-0.d.) syringe needle (Type 304 stainless steel, SGE, Austin, TX) held at 3-3.5 kV as shown in the setup in Figure la. T w o additional continuous-infusion E§ systems were employed in which the analyte solutions were sprayed from the tip of a fused-silica capillary (100-pmi.d., 350pm o.d., SGE) instead of the stainless steel needle. The same syringe pump system was used to pump the analyte solutions (27) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62,1284. (28) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L.; Henion, J. D.; Huang, E. C. Anal. Chem. 1991,63,375.
through these systems. In these cases, connection to the high voltage was made through a metal connector placed several centimeters upstream. The setup in Figure l b employed a stainless steel no-dead-volume fitting (Valco, Houston, TX) to connect the Teflon transfer line to the fused silica via a butt connection. The high voltage was applied to the solution at this connector. With this setup, the area of the metal/solution interfaceat the point of high-voltage contact was minimized. On the basis of the size of this metal fitting and the fused silica, the estimated metal/solution surface area was 10.2 pm2. The system shown in Figure ICemployed the same needle used in the normal ES setup, but the needle was placed upstream. A piece of fused silicawas attached to the needle via a no-dead-volumeconnector. With this setup, the area of the metal/solution interface at the point of high voltage was about equal to that in Figure la, which is estimated to be about 2 pm2. For the flow injection experiments, which were used to judge detection limits, the syringe pump was used to deliver solvent to the needle at a constant rate, through Teflon tubing, to a Rheodyne (Cotati, CA) Model 7520 injector with a 0.5-pL internal sample chamber and then through a short length (ca. 15 cm) of silylated 100-pm4.d. (350-pm-0.d.) silica capillary to which the E§ needle was connected. The metaloctaethylporphyrin samples,prepared from the freebase porphyrins according to standard literature procedures,28 were obtained from Prof. J. Martin E. Quirke (Florida International university). All other analytes were used as obtained from the commercial suppliers as listed anthracene, Fisher Scientific (Fairlawn, NJ); naphthalene, pyrene, and phenothiazine, Chem Service (West Chester, PA); octaethylporphyrin, 2,3-benzanthracene, 9,10-diphenylanthracene, rubrene, and N,N,W,W-tetramethy1-1,4-phenylenediamine(TMPD)Aldrich Chemicals (Milwaukee,WI). Stock solutions of the analyteswere prepared by dissolution of small amounts (ca. 1-3 mg) of each in 10 mL of HPLC grade methanol or methylene chloride (J.T. Baker,Phillipsburg,NJ). The methylenechloride was dried prior to use by elution through a bed of activated basic alumina (BioRad, Richmond, CA) and then stored over alumina. The various analyte/solvent combinations investigated were prepared from these stock solutions to give final analyte concentrations of ca. 1-150 pmol pL-l. In some cases HPLC grade trifluoroacetic acid (J.T. Baker) was added to the solution just prior to analysis at a concentration of 0.05-0.1 % by volume. The amount of material
1588
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
Table I. Summary of Representative Data cod class cDd porphyrins free-baseOEP Mgn(OEP) Nin(OEP) Cun(OEP) Znn(0EP) PAHs naphthalene anthracene
formula
pyrene
2,3-benzanthracene perylene
aromatic amines heterocuomatics
9,lO-diphenylanthracene rubrene TMPD phenothiazine
MW
IE (eV)
534
6.32d 6.19 6.3ad 6.31d
556 590 595 596 128 178 202 228 252 330
532 164 199
6.29" 8.14f 7.45f 7.41f 6.97f 6.90' 6.41f 6.20'
6.96d
E~2m (V)= 0.81e 0.W 0.73O 0.790 0.63e 1.658 1.198 1.258 0.98' 1.04 1.22' 0.82'
1.oOk 0.568
sDecies obsdb (M + H)+ M+ M+ M+ M'+
ndh
M+ M+ M + , pdtd M'+ M'+ M'+ M + , (M + H)+ M'+, (M + H)+
firmre of meritC(fmol) . . 0.8 5 0.5 3 8 I
nd
>lo00 lo00
235 45 70
2.5 3
45
El/zoxvalues, unless otherwise noted, with respect to saturated calomel electrode (SCE).* Species observed depends on solvent system employed in analysis, as discussed in text. The figure of merit, defied for continuous infusion, represents the quality of anal* that paesed out of the capillary needle during the minimum injection period of a single scan of the ion trap necessary to give analyta ion signals 5 times in excess of the standard deviation of the noise. Sample concentrations were 1-100 pmol pL-l and sample flow rates 1-2 pL min-1. Porphyrins were sprayed from solutions composed of methylene chloride/methanoUTFA (10/90/0.1% v/v/v) and the remaining compounds from dry methylene chloride/O.l% TFA. Levin and Lias.29 Fuhrhop et al.u Measured in butyronitrile (0.1 M tetrabutylammonium perchlorate) at platinum electrode. f Lias et 8 Janz and tom kin^.^^ Measured in acetonitrile (0.1 M tetraethylammonium perchlorate) at rotating platinum electrode. nd = not detected. Janz and tom kin^.^^ Peak potentials measured in methylene chloride (0.2 M tetrabutylammonium perchlorate) at platinum-disk electrode. j Addition products formed by nucleophilic reaction of radical cation with certain solvents. k Janz and T0mkins.3~Measured in nitromethane (0.1 M tetraethylammonium perchlorate) at platinum electrode. (I
consumed to acquire the spectra shown, as quoted in the figure headings, corresponds to the amount of analyte which flowed from the ES needle or fused-silica capillary during the data acquisition period.
RESULTS AND DISCUSSION The four ES mass spectra shown in Figure 2, acquired for four different species from four different compound classes (viz., porphyrins, aromatic amines, polycyclic aromatic hydrocarbons (PAHs), and heteroaromatics), demonstrate the ability to produce and detect radical cations from a variety of compound types using a typical ES setup (see Figure la) and the appropriate solvents. Table I presents a representative list of compounds from these different compounds classes for which we have observed radical cations in ES mass spectra. As these data show, generation of radical cations via ES is not an isolated occurrence, but a more general phenomenon with the potential to expand the range of compounds amenable to ESMS and also to expand the types of molecular species that can be generated from particular analytes. Observation of molecular ions in ES mass spectra, as opposed to cationized species, is atypical and was unexpected when first noted with alkyl-substituted metalloporphyrins.19 Although the specific mechanism of formation was unclear,the observation of porphyrin molecular radical cations was rationalized on the basis of the known solution chemistry of metalloporphyrins. One might expect, for example, formation of the protonated metalloporphyrin to be relatively unfavorable when compared to protonation of the free base. This is because the solution-phase basicity of an alkylsubstituted metalloporphyrin is significantly less than that of the corresponding free-base porphyrin.26 In addition, porphyrin radical cations can be formed readily in solution by electrochemical or chemical means, and the radical cations of metalloporphyrins are much more stable in solution than those of the free bases.2+26 Thus, it would be expected that if electron transfer leading to formation of the molecular ion (29)Levin, R. D.;Lias, S.G. Ionization Potential and Appearance PotentiaZMeasurements, 1971-1981. U S . Government Printing Office: Washington, DC, 1982. (30)Lias,S.G.; Bartmess, J. E.; Liebman, J. F.; Holmes, J. L.; Levin, R. D.; Mallard,W. G. Gas-Phase Ion and Neutral Thermochemistry.J. Phys. Chem. Ref.Data 1988,17 (Suppl. 1). (31)Jam,G.J.;T o m b s , R. P. T.NonaqueousEZectroZytesHandbook; Academic Press: New York, 1973;Vol. 11.
is possible in the ES process, it would compete more favorably with proton transfer for the metalloporphyrins than for the free bases, which is what we observed (Le., free-base porphyrins were observed only as (M + H)+).19 A particularly important observation in our study of the origin of radical cations in ES is the effect of the solvent system. The solvent system can play a major role in the types of ions, e.g., M + versu (M + HI+, formed from the anal@. Under solvent conditions other than those noted in Figure 2c,d (i.e., methylene chloride/O.l% TFA), the protonated molecule appears as the major ion in the respective ES maee spectra of N,IVJ',"-tetramethyl-1,4-phenylenediamine (TMPD) and phenothiazine, rather than the radical cation. For example, the protonated molecule is the base peak in the spectrum of TMPD when the compound is sprayed from a solution composed of methylene chloride/methanol/acetic acid (50/50/0.5% v/v/v).l* (NotethatthespectrumofTMPD in Figure 2c shows a considerable abundance of (M + H)+ (mlz 165)as well as the radical cation (mlz 164). On the basis of isotopic abundances,the isotope peak at m/z 165is expected to be only about 125% of the abundance of the radical cation. The remaining contribution to the abundance of the peak at m/z 165is from (M + H)+. This is confirmed by the presence of the (M + H - 15)+fragment ion at m/z 150 resulting from dissociation induced upon injection of (M + H)+ into the trap.19 Possible origins of (M + HI+ in this case are discussed in more detail below.) As another example, the spectrum of 2,3-benzanthracene in Figure 2b exhibits only the radical cation. When this compound was sprayed from a solvent system containing methanol, as will be shown below, ions corresponding in mass to producta of solvent addition reactions were observed. In the case of magnesium octaethylporphyrin (Mg(OEP),Figure 2a) and most other metal(11) porphyrins, the radical cation was the only molecular species observed in a wide range of solvents.'g However, addition of trifluoroacetic acid to the ES solvent was found to demetalate some porphyrins, including Mg(0EP) and Zn(OEP), resulting in observation of a molecular species corresponding to (M - metal + 3H)+. As mentioned above, two mechanisms for formation of radical cations using ES ionization appear to be most probable, viz. a chemical oxidation of the analyk in the solvent prior to or during the spraying process or an electrochemical oxidation in the electrospray needle. In fact, we have already
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
1588
M+ 5561
18741
li
575
0
mh
mlz
lo7i I
(b) (228
520
560
580
600
F l g m 3. Electrospray mass spectra of NYOEP) in (a) methylene chkride/methanoi(QO/lOv/v) and (b) methylene chlorkle/methanoi/ TFA(90/10/0.05%v/v/v)obtainedbyspraylngtherespecthresokRions using the ES setup shown in Figure la.
13071
I 100
540
miz
mh
(M+H-15)+
120
1
140 mh
463/
0
160 mh
180
i:
260
Flgun2. Electrospray massspectraof fouanalytesfromfowdlfferent compound classes in which the molecular radical catlon is observed as the major molecular species. (a)Mg(0EP)sprayed from methylene chloride/methand (90/10 v/v). Approxlmtely 1.0 pmoi of analyte was consumed to acquire the spectrum. (b) 2,3-Benranthracene sprayed from methylene chloride/O. 1% TFA. Approximately 28 pmol of anatytewere consumed to acquirethe spectrum. (c)TMPD sprayed from methylene chlorlde/O.l% TFA. Approximately 15 pmoi of anatyte were consumed to acquire the spectrum. (d) Phenothiazine sprayed from methylene ch&ride/O. 1% TFA. Approximately 29 pmd of analyte were consumed to acquke the spectrum. The ES setup shown in Figure la was used to acquire the spectra.
shown in a separate paperlathat a chemical process, involving reactions between the analyte and ES solvent components, can result in molecular cation and molecular anion formation. A chemical mechanism for radical cation formation in the present case is precluded, however, by the data presented
below. Instead, these data implicats electrochemical oxidation of the analyte at the metal/solution interface of the electrospray needle or at the point of high-voltage contact with the solution as the formation mechanism. Two ES setups, shown in Figure la,b, were employedtoexplore the importance of source geometry in formation of radical cations. If formation of radical cations results from solution chemistry, independent of the ES process, then the physical setup of the ES source should not affect the spectra. If, however, reactions at the metal/solution interface at the point of high-voltage contact are important, as would be the case for an electrochemical mechanism, the spectra might change as the setup is altered. The setup shown in Figure l a is a typical ES source in which the solution is sprayed from a stainless steel needle held at high potential relative to the front aperture of the mass spectrometer. In the setup shown in Figure lb, the solution is sprayed from a fused-silica capillary with the high voltage applied to the solution several centimeters upstream via an in-line metal connector. The most relevant differences between these two spray systems are the metal/solution interface area (ca. 0.2 pm2 (Figure lb) versus 2 pm2 (Figure la)) and the delay between the time when an analyte in solution passes the point of high-voltage contact and when it is sprayed. The solution flow rate was 2 I.~L min-l for both systems. The fused silica attached to the metal connector in Figure l b was 5 cm long so that, at 2 pL min-', the time delay was about 12 8. Both ES setups perform similarly in the analysis of analytes known to be preformed ions in solution (e.g., organic salts such as tetrabutylammonium iodide). However, differences in the spectra obtained with the two systems are apparent when compoundsthat are not necessarily charged in solution, such as the metalloporphyrinsand PAH's, are analyzed. The ES mass spectra shown in Figures 3and 4 were obtained for Ni(0EP) sprayed from each of the two ES setups using two different solvent systems. The spectrum in Figure 3a was obtained by sprayinga methylenechloridelmethanol(101 90 v/v) solution of Ni(0EP) from a stainless steel needle (Figure la). The molecular radical cations, distributed in
is90
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
li41
(a)
I"
10 027 520
0
540
mlz M+ 1590 (M-15)'
1
mh Figure 4. Electrospray mass spectra of NYOEP) in (a) methylene chloride/methanoi(QO/lOv/v) and (b) methylene chkride/methand/ TFA(90/10/0.05% v/v/v)obtained bysprayingthe respecthresolutlons using the ES setup shown in Figure lb.
abundance according to the isotopic composition, are the major ions in the spectrum along with several injectioninduced fragment ions of lower intensity. (Note that the instrument was tuned to induce a significant amount of fragmentation upon injection to identify at a glance the molecular species, M + or (M + H)+,present. We have shownlg that the major fragment ion from the protonated molecule is (M H - 29)+whereas the major fragment from the radical cation is (M - 15)+.) While a solvent system composed of various combinationsof methylene chloride/methanol (from 10190to 90/10 v/v, respectively) worked well in the production of porphyrin radical cations, it was found that radical cation abundance could be increased by addition of trifluoroacetic acid (TFA) to these solvent systems. A comparison of the Ni(0EP) spectra in Figure 3a (methylene chloride/methanol 10/90v/v) and Figure 3b (methylenechloride/methanoVTFA 10/90/0.05% v/v/v) shows that the signal for M + increases by about a factor of 2 upon the addition of TFA. Somewhat different results were obtained when the porphyrin was sprayed from the fused-silica capillary. As shown by the spectrum in Figure 4a, no porphyrin ions were observed in the ES mass spectrum obtained by spraying the methylene chloride/methanol (10/90 v/v) solution of Ni(0EP) from a fused-silica capillary. However,when the methylene chloride/ methanoVTFA (10/90/0.05 5% v/v/v) solution of Ni(0EP) was sprayed from this setup (Figure 4b), the same ions noted when this compound was sprayed from the needle were detected, although at roughly 1 order of magnitude lower abundance. (As will be shown below, the metal/solution interface is involved in M + formation. Therefore, at least some of this difference in abundance is due to the difference in the interface area of the two setups.) Similar results were obtained with the PAH perylene as the analyte in these experiments. Several conclusions can be drawn from the data presented in Figures 3 and 4. First, since the radical cation of Ni(0EP) is not observed when the methylene chloride/methanol solution is sprayed from the fused-silica setup, these ions are not performed in solution. Preformed ions would be observed when spraying is from either setup. Second, the addition of
+
20 053
30 118
40 144
" ' " , " ' ' +
50 '210
60 235
70 Scan 3 0 1 Time
Flgure 5. Total ion current proflle and extracted ion current proflle for the radical cation of NYOEP) (m/z 590) obtained by spraying a methybne chkr#e/methand/TFA (90/10/0.05 % v/v/v) sdutbn of NYOEP) using the ES spray system illustrated in Figure IC. A 30-cm length of 100-pm4.d.fused-siiica capillary was attached tothe end of the needle. The NYOEP) solution was pumped through the system at a rate of 2 pL min-I, for several minutes, with the high voltage turned off. The high voltage was then turned on and spectra acquisbn started. The time delay before the appearance of M'+ is approximately equal to the calculated time to elute the void-volume from the fused silica/ needle butt connection to the spray end of the silica.
TFA to the analyte solution enhancesthe signal for the radical cation in both ES setups, but the mechanism for this enhancement ia not clear. And third, best signals for the radical cation are obtained when spraying is from the stainless steel needle. To more fully understand the differences among the spectra acquired using the different spray setups and solvent systems and to c o n f i i that TFA was not involved in a chemical formation of the radical cations observed, the following experiment was carried out using the ES spray system illustrated in Figure IC.The stainless steel ES needle and metal low-dead-volume connector used in the ES setups in Figure la,b were placed upstream such that the metaVsolution interface area was about equal to that in the ES setup shown in Figure la. Attached to the end of the needle were variable lengths (15,30, and 60 cm) of 100-pm4.d. (350-pm4.d.) fusedsilica capillary. A solution of the analyte of interest was pumped through the system at a rate of 2 p L min-', for several minutes, with the high voltage turned off. The high voltage was then turned on and data acquisition started. For compounds that are preformed ions in solution, like tetrabutylammonium iodide, signal due to the molecular species is observed essentially immediately upon turning on the high voltage. The observation is different, however, for neutral analytes. In the case of Ni(OEP1, dissolved in methylene chloride/methanol/TFA (10/90/0.05% v/v/v), the porphyrin radical cation (mlz 590) was not observed in the early stages of data acquisition. The time delay before the appearance of M + was approximatelyequal to the time to elute the voidvolume from the fused silicaheedle butt connection to the spray end of the silica, which calculated (for a solution flow of 2 p L min-1) to be about 36,72, and 141 s, for the 15-, 30-, and 60-cm sections of silica, respectively. As an example, the total ion current profile and extracted ion current profile for the ion at m/z 590, acquired in the experiment using the 30cm length of silica, are shown in Figure 5. A steady total ion current (Figure 5a) is noted until 85 8, the approximate time calculated for elution of the void-volume, at which point a significant increase, over a short period of time, to a new steady-state ion current occurs. The extracted ion current for the radical cation of Ni(0EP) (Figure 5b, m/z 590) shows that this increase in ion current coincidee with the appearance of the radical cation. Before elution of the void-volume (scan 18, Figure 6a) a low-abundance peak corresponding to protonated Ni(0EP) (mlz 591) is observed along with its diagnostic fragment ion at m/z 562. This species is probably formed in solution, albeit inefficiently, by protonation by TFA since it is not observed in experiments conducted without
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
(M+H)+ 591
I
562
1
M+ 590
100
575
(b)
l~
mh
Flgure 6. Electrospray mass spectra obtained at (a) scan 18 and (b) scan 44 in the ion current profiles shown in Flgure 5.
the acid. After elution of the void-volume (scan 44, Figure 6b), the radical cation and expected injection-induced fragments are observed in the spectrum. Once again these data clearly indicate that the metalloporphyrin radical cations are not preformed ions in solution. Moreover, the metal/solution interface in the ES setup is certainly involved in their formation. The delay period from turning on the high voltage to first observation of the radical cation coincides with the time necessary to elute the fiist portion of the analyte sample that was at the metal/solution interface when the high voltage was turned on. We believe that at the metal/solution interface a conventional electrochemical oxidation of the analyte takes place. An electrochemical mechanism for formation of the radical cations in ES is consistent with the recent description of the ES source by Kebarle and c ~ - w o r k e r s ~ J ~ "electrolysis cell of a as~an special kind". In a typical ES source, highly charged droplets of a solution containing the analyte are dispersed at atmospheric pressure through application of a high potential difference (typically 3-5 kV of the same polarity as the ion of interest) between a stainless steel capillary needle, through which the analyte solution is flowing, and the atmospheric sampling aperture of the mass spectrometer. As described by Kebarle and c o - ~ o r k e r s ~ and ~ Jothers,ll ~ in the positiveion mode of operation, a partial separation of positive and negative ions present in the solution occurs leading to an excess of positive charges on the surface of the liquid at the needle tip. This excess charge destabilizes the surface and leads to emission of positively charged droplets from the needle tip. This is followed by droplet evaporation and finally ion evaporation or desorption to yield gas-phase ions that can be sampled and analyzed by the mass spectrometer. Important to the present work is the charge separation process, termed electrophoreticcharge separation, deemed responsible for the generation of a charged solution at the needle tip and subsequent electrospray. A significant aspect of the work by Kebarle and ~ o - w o r k e r s was ~ ~ Jthe ~ recognition (and subsequent experimental verification) that if the electrophoretic charge separation mechanism of ES was operational, charge balance required a flow of electrons into the needle to compensate for the positive charge sprayed from the needle. In other words, a conventional electrochemical oxidation reaction must occur at the metal/solution interface of the
1191
needle. A given set of conditions (e.g., solvent(s), flow rates, applied voltages, needle dimensions, etc.) should, within certain limits, determinethe potential gradients possible along the metal/solution interface. The particular oxidation reaction(s) that occur to balance the charge flow will depend on the magnitude of these gradients and on the relative electrochemicaloxidation potentials of the various species in solution and their relative concentrations. In aqueous or "wet" solutions, an example used by Kebarle and co-~orkers,12J3 oxidation of hydroxide anions or water, reactions which require potentials of less than 1 V, might be the source of electrons. Types of Analytes Observed as M*+.In light of an electrochemical mechanism for radical cation formation, observation of the compounds listed in Table I, and compounds with similar electrochemicalbehavior, as M + in ES mass spectra might be expected. For example, facile electrochemical oxidation of compounds in each of the four compound classes represented in Table I is well documented21-26p32-35 and the radical cations formed are often stable in solution. In fact, in some cases, the radical cations formed are so stable that their salts can be isolated. Among the most stable and most studied of these salts are metalloporphyrin perchlorates and perchlorate salts of diaminobenzenes such as TMPD (i.e., Wurster salts)." The Compounds listed in Table I, and other molecules typically easy to oxidize, are composed of highly conjugated systems and/or contain heteroatoms with lone pair electrons. These structural characteristics aid in delocalization of the unpaired electron and positive charge, thereby stabilizing the radical cation. Substitution of these molecules with electrondonating groups, e.g., -OH, -OCHa, -N(CH&, and 4 H 3 , can also aid in ion stabilization." In more fundamental terms, the ability of a neutral organic molecule to give up an electron (i.e., to be oxidized) is governed by the energy of its highest occupied molecular orbital (HOMO),which can be calculated approximately or can be estimated experimentally by measurements of the half-wave potentials for solution-phase oxidation (Elpox)or gas-phase ionization energies (E's)." Because HOMO energies govern both Elpoxvalues and IE's, it is expected, and has been found, that gas-phase IE's and values correlate over a broad range of compound types.% However, solvation phenomena affect Elpoxvalues so that best correlations are made using E1poxvalues measured under identical experimental condition^.^^ The Elpoxvalues and gas-phase IE's listed for the compounds in Table I show the expected correlation even though the tabulated El/zo.values were obtained under several sets of conditions. The most easily oxidized compound in this list, on the basis of Elpox values, is Mg(0EP) (Elpox= 0.54 V), while the most difficult to oxidize is naphthalene (Elpox= 1.65 V). The IE's, which increase as the molecule becomes more difficult to ionize, show the same general trend. The present data might be used to predict whether a compound will form a radical cation in ES. Excluding freebase OEP, which formed (M + HI+, only one compound listed, viz. naphthalene, could not be detected as the radical cation. The IE's and Elpoxvalues for this compound, may therefore, represent upper limits for formation of M'+ in ES under the (32) Peover, M. E. In Electroanalytical Chemistry; Bard, A. J., Ed.; M. Dekker: New York, 1967; Vol. 2, pp 1-51. (33) Eberson, L.;Nyberg, K. In Aduances in Physical Organic Chemistry; Gold, V., Bethell, D., Eds.; Academic Press: New York, 1976; Vol. 12, pp 1-129. (34) Bard, A. J.; Ledwith, A.; Shine, H. J. In Advances in Physical Organic Chemistry; Gold, V., Bethell, D., Eds.; Academic Press: New York, 1976; Vol. 13, p 155-278. (35)Yoshida, K. Electrooxidation in Organic Chemktry;John Wiley: New York, 1984. (36) Miller, L.L.;Nordblom, G. d.; Mayeda, E. A. J.Org. Chem. 1972, 7, 916.
1592
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
current experimental conditions. If this is so, the potential gradient at the metal/solution interface that drives the electron-transfer process must range up to only about 1.5V. Compounds with Elpoxvalues above 1.5 V and IEs greater than about 8.0 eV would probably not, therefore, form radical cations. These conclusions are supported by the fact that the relative ionization efficienciesfor these compounds (based on the figures of merit in Table I) also decrease as the IE’s and E1/zOx values increase, possibly indicating less efficient formation of the radical cation as the potential gradient at the metal/solution interface approaches the E1porvalue for the reaction. Role of the Solvent System in Observing Analytes as M*+. The ability to oxidize the analyte in solution is only one factor in the observation of radical cations in ES mass spectra. As already discussed, free-base OEP, although easily oxidized in solution, is observed as the protonated molecule and the molecular species observed in the ES mass spectra of TMPD and phenothiazine depends on the ES solvent system. Furthermore, data obtained with spraying from the fusedsilica system demonstrated that M*+ions of Ni(0EP) could not be observed without the addition of TFA to the solvent system. These results clearly indicate that the ES solvent is a very important parameter in the production or preservation of radical cations in ES. Typical solvent systems for ESMS are composed of various combinations of methanol, acetonitrile, and/or water along with a small amount of acidic or basic additive^.^-^ Such solvent systems are chosen because of the solubility characteristics of the more common analytes (e.g., peptides and proteins), because they produce a stable spray and because they allow for solution-phase ionization of the compounds (typically ionization via salt dissolution or acid/base chemistry). Observationof radical cations in ESMS must involve a more careful selection of solvents because radical cations can be consumed by several types of rapid reactions in solution.32-35 Particularly important, for example, are nucleophilic reactions with solvents or solvent additives. Electrochemists have been able to avoid or minimize these reactions, thereby ”stabilizing”and extending the lifetime of radical cations in solution, through the judicious choice of solvents and solvent additives. Protic solvents as well as nucleophilic solvents (e.g., water and methanol) and nucleophilic solvent additives (e.g., acetate anion) are typically avoided. Aprotic, nonnucleophilic solvents such as dried acetonitrile and dried methylene chloride are commonly employed in electrochemical generation of radical cations. Interestingly, the use of TFA as the solvent37or as a solvent additive (e.g., TFA in methylene ~ h l o r i d e ~ sin . ~ ~electro) chemical generation of radical cations has been found to “stabilize” the ions. DannenbereO has shown, on the basis of molecular orbital calculations,that this stabilization occurs may means of interactions between the nonnucleophilic CF3 group of TFA and the cation. Attack of the TFA-solvated (i.e., stabilized) radical cation by nucleophilic species is in this way hindered. The facts that the lifetimes of radical cations in solution are limited, as a result of rapid solution reactions, and that TFA stabilizes radical cations in solution help to explain the results presented in Figures 3 and 4. The spectra in Figure 4 were recorded by spraying a methylene chloride/methanol (10/90 v/v) solution of Ni(0EP) from the fused-silica system shown in Figure lb. With this system there was a 12-5 delay time between formation of the radical cation at the upstream (37) Hammerich, 0.;Moe, N. S.; Parker, V. D. J. Chem. Soc., Chem. Common. 1974, 33. (38) Bechgaard, K.; Parker, V. D. J. Am. Chem. SOC.1972,94,4749. (39) Ronlan, A.; Parker, V. D. J. Chem. SOC.,Chem. Commun. 1974, 33. (40) Dannenberg, J. J. Angew. Chem., Int. E d . Engl. 1976, 14, 641.
metal connector and spraying of the ion from the 5-cm length of fused silica. Apparently, the lifetime of the radical cation in this situation was less than the delay time, and therefore, it was not detected (Figure 4a). Upon addition of TFA to the solution, M + was observed (Figure 4b). The TFA probably acts to stabilize the radical cation, Le., slow the kinetics of radical cation loss, thereby extending its solution lifetime beyond the 12-5 delay time. Similar reasoning explains the enhancement in radical cation abundance upon addition of TFA to the solvent when spraying is from the needle system (Figure 3). In this case, the radical cation of Ni(0EP) can be observed even without the addition of TFA (Figure 3a). This is possible because radical cations can be produced along the entire length of the needle. Even very short-lived radical cations can be detected in such a case because they can be made at the needle tip just as the solution is sprayed. The M + signal is enhanced upon the addition of TFA, however, because those radical cations produced farthest upstream in the needle, which might otherwise react away, are stabilized and detected adding to the overall signal. Other evidence for the need to protect radical cations of certain molecules from solution reactions is also evident in our data. For example, a significant (M+ H)+ion was noted in the spectrum of TMPD in Figure 2c. While it might be argued that this results from protonationof TMPD in solution, Russel141has shown in electrochemical experiments that this ion can result from reduction of the TMPD radical cation by traces of water present in the solvent (eq 1).A similar reaction 2TMPD”
+ H,O
-
2(TMPD + H)’+ 1/20, (1)
might be responsible for the observation of free-base OEP as the protonated molecule. Free-base porphyrins are known to be easily oxidized to form radical cations in solution but are much less stable than the corresponding metal derivatives.26 A reaction sequence might therefore be conceived in which the radical cation generated in the ES process undergoes a rapid reaction with a solvent species (probably methanol or water) to form (M + H)+. More explicit evidence of solution reactions comes from a comparison of ESMS results for the PAH’s perylene and 2,3benzanthracene. The spectra obtained when both these compounds are sprayed from methylene chloride/methanol (10/90v/v), using the ES setup in Figure la, are shown in Figure 7. The spectrum of perylene (Figure 7a) shows only the radical cation while that of 2,3-benzanthracene (Figure 7b) contains a number of abundant peaks in the molecular ion region. The latter spectrum can be contrasted with that shown in Figure 2b which was obtained by spraying 2,3-benzanthracenefrom dry methylenechloride/O.l% TFA. In that case only the radical cation was observed. Although not explicitly identified here, these peaks in the molecular ion region of the spectrum in Figure 7b undoubtedly arise from reaction of the radical cation with species in the solventa (probably methanol and/or water). The spectrum in Figure 2b does not show these ions since the reactive species are absent from the solvent system. These results are consistent with the observations of Phelps et al.42and Marcoux et al.43 with electrochemicallygenerated PAH radical cations. These workers found that whether or not compounds of this type underwent rapid followup reactions in solution depended on whether they were substituted at the positions of high electron density. Radical cations of substituted PAHs such as 9,lOdiphenylanthracene, perylene, and rubrene were found to be much more stable in solution than unsubstituted ones such (41) Russell, C. D. Anul. Chem. 1963,36, 1291. (42) Phelps, J.; Santhanam, K. S. V.; Bard, A. J. J . Am. Chem. SOC. 1967,89, 1752. (43) Marcoux, L. S.; Fritsch, J. M.; Adams, R. N. J. Am. Chem. SOC. 1967,89, 5766.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992 8.45 pmol
M+
1001
252
1589
11 I
1001 ~
4.2 pmol
,I
I
I l l
il 2
0 mh
600 32:21
1
1200 21:31
1800 32:21
2400 Scan 43:lO Time
Flgurr 8. Extractedlon current profile for the combined radlcai catbns of NYOEP) (mlz 590-593) obtained from a flow injection expewiment in which replicate injectlons of a blank solution and analyte eolutions of increasing concentration were made. Ni(OEP) was dissolved in methylene chloride/methanoi/TFA (lO/QO/O. 1% v/v/v) and injected into a solvent stream of the same compositlonflowing at 1.5 p l mln-'
.
down to at least 420 fmol injected. A similar experiment for rubrene found this compound detectable down to at least 640 fmol. 2 mh
Flgwe 7. Electrospray mass spectra of (a) perylene and (b) 2,s benzanthraceneboth sprayed from methylene chloride/methanol(1O/ 90 v/v) using the ES setup shown In Figure la.
as anthracene and 2,3-benzanthracenea This observation also points to another possible reason why the radical cation of naphthalene could not be observed in our experiments and why the relative ionization efficiencies of anthracene and pyrene (based on the figures of merit in Table I) were so low. Even if the radical cation of these species is produced by the ES process, it may be so reactive that followup reactions, even in carefully prepared solutions, consume the ion before it can be detected. On the basis of data and discussion above it is apparent that in order to observe a wide range of analytes as radical cations in ES and to enhance the abundance of these ions, solvents and solvent additives that can stabilize radical cations and/or minimize chemical followup reactions, in addition to producing a stable ES, must be used in the analysis. Furthermore, minimizing the time from radical cation formation to detection, as is done with spraying from a metal needle (Figure la), should increase the abundance of the analyte ion, since the opportunity for followup reactions will be reduced. Analytical Applications. The ability to generate radical cations via the ES process clearly has significant potential in analytical applications. First of all, ionization via electron transfer opens to ESMS the analysis of compound types not typically amenable to the technique. These are neutral, nonpolar compounds,such as the PAH's, that cannot be charged by conventional solution-phase acidlbase chemistry. Also, molecular species other than the normal molecular adduct ions can be produced for selected types of compounds. In the best cases, such as for the metalloporphyrins and relatively unreactive PAHs such as perylene and rubrene, the levels of detection for the ES-produced radical cations, based on the figures of merit listed in Table I and on the results of flow injection experiments (see below), are comparable to those levels we have obtained on our instrument with preformed ionic species (see e.g., refs 19, 27, and 28). As an example, Figure 8 shows the extracted ion current for the combined molecular radical cations (mlz 590-593) of Ni(0EP) obtained in a flow injection experiment in which replicate injections of a blank solution and analyte solutions of increasing concentration were made. Clearly, Ni(0EP) is detectable
For compounds amenable to electrochemicaloxidation in solution,the greatest challenge to utilization of this ionization phenomenon in analytical applications appears to be constraints placed on the solvent systems to minimize radical cation/solventreactions that would interfere with the analysis. The best solvent systems for producing and preserving the radical cations (e.g., methylene chloride/O.l % TFA) are more difficult to spray than a solvent composed largely of methanol, for example. While this limitation is minor in continuousinfusion or flow injection experiments,the solvent constraints will more severely impact analyses where on-line separations are involved. The data in this paper also indicate that the potential gradients at the metal/solution interface might be limited under the present conditions to about 1.5 V. Such a limit in potential gradient will confine the range of compounds that can be oxidized to form radical cations. Changing solvent flow rates, needle dimensions (and possibly needle material), and applied voltages, for example, might be explored as ways to extend these potential gradients. One method we have explored to overcome this possible gradient limit is incorporation of an electrochemicalcell, normally used as an HPLC electrochemical detector, into the ES system rather than a needle or other metal high-voltage contact.44 Initial results with this setup demonstrated some degree of control over the oxidation reactions and analyte ions observed, suggestingthat additional control over potential gradients at the met& solution interface is possible.
ACKNOWLEDGMENT We thank J. Martin E. Quirke (Florida International University) for supplying the metal octaethylporphyrin samples and Ted R.Mueller (ORNL) for helpful discussions regarding the electrochemical aspects of this work. This research was sponsored by the United State Department of Energy, Office of Basic Energy Sciences, under Contract DEAC05-840R21400with Martin Marietta Energy Systems,Inc.
RECEIVED for review January 7, 1992. Accepted April 20, 1992. (44)Van Berkel, G. J.; McLuckey, S. A; Glish, G. L. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 19-24,1991;ASMS East Lansing, MI; p 1237.
