Anal. Chem. 1995, 67,2892-2900
Mechanistic Interpretation of the Dependence of Charge State Distributions on Analyte Concentrations in Electrospray Ionization Mass Spectrometry Guangdi Wang and Richard B. Cole*
Department of Chemistry, University of New Orleans, Lakefronf, New Orleans, Louisiana 70148
The effect of analyte concentrations upon charge state distributions in electrospray ionization mass spectrometry (ESMS) was investigated using four compounds which showed doubly charged and singly charged positive ions (gramicidin S, 4,4‘-bipiperidine), multiply charged negative ions (Trypan Blue), or doubly charged and singly charged negative ions (eosin Y) in ES mass spectra. For each of the above compounds, the charge associated with analyte molecules was calculated to increase in solution with increasing concentration,yet the analyte charge state distributions in ES mass spectra were observed to monotonically shift toward lower values. In order to rationalize these observations,we introduce the ratio N/No,the ratio of the number of excess charges on all droplets produced by electrospray (N) to the total number of analyte molecules in all droplets (No). For each compound, the N/No value decreases with increasing analyte concentration. The lower N/No value is proposed to be an underlying factor critical to explaining the desorption of a higher proportion of gas-phase ions of lower charge state at elevated analyte concentrations. We propose that the lower N/No value is indicative of a decreased efficiency of analyte charging and an increased level of ion pairing of charged analyte molecules with available counterions. Furthermore, in comparing N/No values and observed analyte charge state distributions as functions of increasing analyte concentration, the decreasing N/No ratio predicts a much greater extent of shifting of analyte charge states toward lower values than was observed. This implies that the actual degree of ion pairing in droplets was considerablyalleviated as compared to that indicated by the calculated N / N o values. This finding conforms to a description of the ES process wherein uneven fission of droplets occurs at the Rayleigh limit,thereby generating offspring droplets of higher charge to mass ratio, hence an augmented N / N o value. In electrospray ionization mass spectrometry (ESMS), solutions containing analytes in ionic form (either singly or multiply charged) are analyzed rather routinely. Ions are most often formed from neutral analytes via protonation/cation attachment in the positive ion modele3 or via dissociation of protons or other ~
~
~~
(1) Thomson, B A, Inbame, J V IDziedzic, P J Anal Chem 1982,54,22192224
2892 Analytml ChemWy, Vol. 67, No 17, September 1, 1995
cations in the negative ion There are different situations, however, where the detected ions can be formed as a result of electron transfer processes (either direct or indirect) at the metal-liquid interface of the capillary,8-10as the electrospray mass spectrometer can be viewed as a special type of electrochemical cell.” Furthermore, previous reports12-14 have shown that ions which are virtually nonexistent in bulk solution (e.g., multiply protonated molecules at high pH) may be generated during the electrospray process. Relative to the number of recent literature reports describing applications of ESMS to structural and identification problems involving both large and not so large polar molecules (e.g., 100100 000 Da), the number of reports investigating fundamental aspects of the ionization mechanism have been fewer. Existing models depicting ES ionization processes include the charged residue model (CRM) originally described by Dole et and the ion evaporation model OEM) first proposed by Iribame and Thomson.16J7 These models describe events that occur after an electrostatic field is applied to a liquid that is being forced (pressure) through a metal capillary. Charged droplets are emitted from a ‘Taylor cone”, which forms in response to the imposed electric field. Evaporation of solvent contained in the droplets is critical to the ultimate formation of gas-phase ions in both cases. The two models differ in the description of events occurring in the later stage($ of the droplet lifetime. Parentheti(2) Iribarne, J. V.;Dziedzic, P. J.; Thomson, B. A. Int. J. Mass Spectrom. Ion Phys. 1 9 8 3 , 50, 331-334. (3) Whitehouse, C. M.; Dreyer. R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675-679. (4) Covey, T. R.: Bruins. A. P.; Henion, J. D. Org. Muss Spectrom. 1988, 23, 178-186. (5) Edmonds, C. G.; Loo, J. A; Barinaga, C. J.: Udseth, H. R.; Smith, R D. J. Chromatogr. 1989, 474, 21-37. (6) Smith, R. D.; Loo, J. A: Edmonds, C. G.; Barinaga, C. J.: Udseth, H. R. Anal. Chem. 1 9 9 0 , 62, 882-899. (7) Cole, R B.; Hamata, A. K. J. Am. SOC.Mass Spectrom. 1 9 9 3 , 4, 546-556. (8) Van Berkel, G. J.; McLuckey, S. A,: Glish, G. L. Anal. Chem. 1991, 63, 1098- 1109. (9) Van Berkel, G. J.; McLuckey, S. A,; Glish, G. L. Anal. Chem. 1 9 9 2 , 64, 1586-1593. (10) Xu, X.; Nolan, S. P.; Cole, R B. Anal. Chem. 1994, 66, 119-125. (11) Blades, A. T.; Ikonomou, M. G.: Kebarle, P. Anal. Chem. 1991,63,21092114. (12) Kelly, M. A: Vestling, M. M.; Fenselau, C. C. Org. Mass Spectrom. 1 9 9 2 , 27, 1143-1147. (13) Gatlin, C. L.: Turecek, F. Anal. Chem. 1994, 66, 712-718. (14) Wang, G.: Cole, R B. Org. Mass Spectrom. 1994, 29. 419-427. (15) Dole. M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (16) Iribarne, J. V.; Thomson, B. A J. Chem. Phys. 1976, 64, 2287-2294. (17) Thomson, B. A; Iribame, J. V.J. Chem. Phys. 1979, 71, 4451-4463. 0003-270019510367-2892$9.00/0 0 1995 American Chemical Society
cally, a model proposing direct production of gas-phase ions at the Taylor cone has also been offered,l8Jg although recent evidence has established that gas-phase ions are produced directly from charged droplets.20 In IEM, preferential evaporation of solvent from the droplet leads to increasing charge densities causing Rayleigh instability and droplet fission, thus spawning offspring droplets with higher charge-to-mass ratios than their precursors. Ion evaporation is proposed to take place when the droplets acquire an electric field large enough to lift the ions into the gas phase. In Dole’s charged residue model, however, droplet fission caused by Rayleigh instability is believed to recur sequentially until the offspring droplets contain only one analyte molecule, which is converted into a gas-phase ion upon evaporation of all solvent molecules. While IEM did not address, when first proposed, the phenomenon of multiple charging, CRM held that the multiple charging phenomenon resulted from excess charges left on the final droplet that contained only a single analyte molecule. In 1993, Fenn21 reported a detailed electrospray ionization model which extended concepts derived from ion evaporation theory in efforts to elucidate the mechanism of formation of multiply charged gas-phase ions from charged droplets. This treatment described the dependence of charge state distributions on a number of parameters such as analyte conformation and solvent evaporation. It also depicted the spacing of charges on the surface of the droplet as being critical to determining the desorption rate and the location of charges on the desorbed ion. More recently, Kebarle and Tangz2 elaborated on a modified charged residue model which they called single ion in a droplet theory (SIDT). This treatment takes into account the uneven fission characteristics of charged droplets which have been observed by several Uneven fission of a shrinking droplet yields a “tail” of numerous substantially smaller offspring droplets which carry about 15%of the parent droplet charge and only 2% of the parent droplet mass.23,25The offspring droplets may also be enriched in surface-active ions, providing a rationalization for selective ion enrichment observed in ESMSZ2 Another distinguishing feature between SIDT and Fenn’s modified ion evaporation model was suggested, namely, that the source of energy required to overcome the activation barrier to creating gas-phase ions from charged droplets is different. In Fenn’s model, the activation energy is thermal, whereas in Kebarle’s model, ion formation is activated by elastic deformation of the droplets. In a previous reportz6 we showed that, for protein molecules and other analytes, charge state distributions observed in ESMS were not significantly affected by an increasing conductivity of solution when external electrolyte (not participating in charge attachment equilibria) was added. The presence of high concentrations of electrolytes very likely changes the distribution of (18) Siu, K. W. M.: Guevremont, R.; Le Blanc, J. C. Y.; O’Brien, R. T.: Berman,
S. S. Org. Mass Spectrom. 1993,28, 579-584. (19) Guevremont, R: Le Blanc, J. C. Y.: Siu, K W. M. 0%.Mass Spectrom. 1993, 28, 134551352, (20) Hager, D. B.: Dovichi, h’.J.: Klassen, J.: Kebarle, P. Anal. Chem. 1994,66. 3944- 3949. (21) Fenn, J. B. J Am. Soc. Mass Spectrom. 1993,4, 524-535. (22) Kebarle, P.: Tang, L. Anal. Chem. 1993,65,972A-986A. (23) Taflin, D. C.: Ward, T. L.: Davis, E. J. Langmuir 1989,5,376-384. (24) Sheehan, E.: Willoughby, R. Proceedings of the 41st ASMS Conference on Mass Spectrom. and Allied Topics, San Francisco, C A 1993; p 770a-b. (25) Gomez. A.: Tang, K. Phys. Fluids 1994,6. 404-414, (26) Wang, G.; Cole, R. B. Anal. Chem. 1994,66. 3702-3708.
excess charges in the droplets, leading to an increasing proportion of electrolyte ions near the droplet surface, hence an increasing suppression of analyte signal intensity. We will show in this study that an increase in solution conductivity brought forth by an increase in analyte concentration (salt form) not only changes the total ion abundance but also considerably shifts the anaiyte charge state distribution in both positive and negative ion electrospray mass spectrometry. These experimental observations have led us to propose a new mechanistic interpretation to rationalize changes in charge state distributions observed in response to varying analyte concentrations in electrosprayed droplets. EXPERIMENTAL SECTION Mass spectrometry experiments were performed on a Vestec201 quadrupole mass spectrometer (Vestec Corp., PerSeptive Biosystems, Houston, TX) equipped with an electrospray ionization source which has been described previou~ly.~~ To minimize the effect of instrumental conditions on charge state distributions, mass spectrometer operating parameters were kept constant from run to run: in the positive ion mode, the applied voltage at the nozzle was 300 V, and the skimmer-collimator voltage difference was 4 V (minimal fragmentation conditions); in the negative ion mode, the corresponding voltages were -280 and -4 V, respectively. The flow rate of sample solutions was 1.6 pL/min throughout the ESMS experiments. The source block temperature was maintained at 256 & 1 “C, and the electrospray needle temperature as indicated by a thermocouple located in the vicinity of the needle tip was 46 f 1 “C (except during the acquisition of the mass spectrum shown in Figure 6, where the block temperature was lowered to 70 “C). Although the electrospray needle voltage was varied slightly between 2.0 and 2.5 kV (positive ion mode) and between -2.0 and -2.5 kV (negative ion mode) to achieve optimal signal intensity and stability, other parameters being constant, the analyte charge states exhibited no discemable shift within the employed range. Total ES ion current was measured at the nozzle counterelectrode. Finally, each series of analyte solutions at different concentrations was run within a period of 2 h. All analytes were capable of forming singly and doubly (or multiply) charged species via protonation or dissociation in methanol solution. 4,4’-Bipiperidine dihydrochloride, and eosin Y were obtained from Aldrich Chemical Co. (St. Louis, MO); gramicidin S and Trypan Blue were purchased from Sigma Chemical Co. (St. Louis, MO). Analyte charge state distributions were evaluated by calculating an average charge state value (2),26 which was defined as follows:
where Z A I -is , ~the ~ signal intensity of analyte ion A in a given charge state detected by the mass spectrometer and i represents the number of charges carried by the ion. Average charge state (2)values were obtained using the signal intensity for each multiply charged ion appearing in ES mass spectra. Because no correction was made for the transmission bias of the quadrupole analyzer, the charge state distributions exhibited by ions in ES mass spectra have a slight systematic bias toward higher values (27) Allen, M. A.; Vestal, M. L. 1.Am. Sot. Mass Spectrom. 1992,3,18-26.
Analytical Chemistry, Vol. 67, No. 1 Z,September I, 7995
2893
as compared to those exhibited by ions in the ion source. For a given analyte, however, this transmission bias will affect the charge states in a constant manner, independent of the analyte concentration. On the other hand, because higher charge state ions are more susceptible to CID processes, this bias will be compensated to some extent by any collision-induced dissociation (CID), which causes a shift in charge states toward lower values. RESULTS AND DISCUSSION Earlier investigations into the behavior of liquids under electrospray conditionsz8~z9 showed that the total ion current follows a weak dependence on the conductivity of the sprayed liquid given as I = Ha", where Hand n are constants (0.2 < n < 0.4) and CT is the liquid conductivity. Solution conductivity can be enhanced either by adding external electrolytes or by increasing the concentration of a dissociating analyte in solution. In both cases, the total ES ion current increases as a result, although the magnitude of the current increase is considerably less than the rise in concentration. We have shownz6 that upon addition of external electrolyte (not participating in charge attachment to/ removal from the analyte), the charge state distributions of analytes do not change significantly despite a dramatic reduction in overall analyte signal response in the presence of increasing electrolyte signal intensity. Increasing the concentration of the analyte itself, present in salt form, represents a different means of influencing solution conductivity that merits a separate treatment. Among four analytes employed in this study, the ES mass spectra of gramicidin S14 and eosin Y 3 O have been reported previously. The molecular structures and ES mass spectra of 4,4'bipiperidine dihydrochloride (4,4'-BPD) and Trypan Blue are shown in Figure 1. 4,4'-BPD (Figure la) shows peaks corresponding to doubly charged ions at m / z 85 and singly charged ions at m / z 169. Trypan Blue (Figure lb) gives a singly charged ion, (M - Na)l-, and several multiply charged ions ranging from @ - 2Na)2I to (M - 4Na)4- as indicated in the figure. In results presented below, we find in both positive and negative ion ESMS that increasing concentrations of these small analyte species (MW ~ 2 0 0 0 resulted ) in consistently lowered charge states in ES mass spectra. These observations are in agreement with previously reported results in positive ion ESMS.6J4~21~31 It is generally believed that the droplet charge is more readily depleted when analyte molecules bearing basic sites are present in increasing concentrations. In Fenn's modelz1it is argued that, at high analyte concentrations, analyte molecules are likely to carry off droplet charges via ion evaporation of low charge state ions at earlier moments in the droplet lifetime, as compared to lower concentration solutions. The current study represents a further examination of the mechanism underlying the concentration effect on charge state distributions. Positive Ion ESMS. Figure 2a shows the abundances of singly charged and doubly charged ions of gramicidin S as functions of concentration. In the lower concentration regime from to 8 x M (first two data points from left), both the doubly charged and the singly charged ion abundances increased at about the same rate; in the intermediate concentration regime (28) F'feifer, R. J; Hendricks. C. D. ALAA J. 1968,6. 496-502. (29) Smith, D. P. H. IEEE Trans. Ind. Appl. 1986,LA-22, 527-535. (30) Varghese, J.; Cole, R. B. J. Chromatogr. 1993,639, 303-316. (31) Chowdhury, S.; Katta,V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4,81-87.
2894 Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
doubly protonated 4,4'-bipiperidine
169 (M+HY
050
100
d z
200
150
b
40001 3500
1
3000 2500 w
1500
446 (M.3Na+H$'
180
500
lab0
mh
Figure 1. Positive ion electrospray mass spectra of (a) 4,4'bipiperidine (4,4'-BPD) and (b) Trypan Blue.
from 8 x to 3 x 10-5 M (third and forth data points from left), the abundance of singly charged ions increased slightly more rapidly. As a result, the average charge state 2 (Figure 2b) began to decrease significantly above 8 x 10+ M. As the concentration of gramicidin S was raised above 1.6 x M (fifth data point from left), both the doubly and singly charged ion abundances began to decrease, with the doubly charged species decreasing to a greater extent, causing a continued lowering of the average charge state in ES mass spectra. The diminishing of overall analyte signal intensity at very high concentrations may be caused by more droplets reaching the solid residual limit before Rayleigh instability and/or ion evaporation occur.16 Shown in Figure 3 are the analogous data obtained for 4 4 BPD. Again,at concentrations up to M (first three data points from left), the signal intensity of both doubly charged and singly charged analyte increased at close to the same rate (Figure 3a) such that the charge state distribution was only lowered slightly (Figure 3b). Above M, however, the average 2 value decreased considerably with increasing analyte concentration. Under constant experimental conditions, the observed changes in mass spectrometrically detected ion current and charge state distributions cannot be attributed to instrumental factors. To explain the observed overall shift of charge state distributions toward lower values with increasing analyte concentrations, we first examine the solution-phase factors which change as the analyte concentration is varied. For gramicidin S and 4,4'-
-
n 'P a
a
1~10~1
d
1+
XlOO
1
'
"""'I
'
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'
"""'I
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'
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21
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.,
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1x10-2 ' """'I ' """'I ' """'I ' """'1 '""."I '""-7 iX10.7 1 ~ 1 0 . 6 1 ~ 1 0 . 5IXIO.* 1 ~ 1 0 . 31 ~ 1 0 . 21 x 1 0 . ~ Concentration (M)
'"''1111
1 ~ 1 0 . 6 1 ~ 1 0 . 5 iX10e4 iX10.' Concentration (M)
1x10.~
Figure 2. Effect of analyte concentration, shown in logarithmic plots, on (a) abundances of singly charged and doubly charged ions, (b) average charge state Z,and (c) NIN, ratio, for 4,4'-bipiperidine.
Figure 3. log-log plots of (a) abundances of singly charged and doubly charged ions, (b) average charge state Z,and (c) N/No ratio vs concentration, for gramicidin S.
+
+
bipiperidine, which were originally obtained in the diprotonated (chloride salt) form, and presuming complete dissociation of chloride counterions initially present, the dissociation of protons in polar solvent may be simply modeled as follows:
MH;+
LL MH+ + H+
MH+
M
+ H+
where M, MH', MHz2+ are neutral, monoprotonated, and diprotonated analyte species, respectively; K,I and Kd are the acid dissociation constants. The concentration ratio of [ M H P I / [MH+] can be expressed as
[MH,2'1/[MH+l = [H+l/K,, In estimating the ratio [MHZ~+I/[MH+I in solution, pH values of gramicidin S and 4,4'-bipiperidine solutions at various concentrations were measured. The pKal values were estimated to be 10.16 for doubly protonated gramicidin S32and 9.5 for doubly protonated 4,4'-bipiperidine in aqueous s0lution.3~Listed in Table (32) Perrin, D. D. Dissociation Constants of Organic Bases in Aqueous Solution; Butterworths: London, 1%5. (33) Gerzon, IC; Cochran, J. E., Jr.; White, L. A; Monahan. R; Krumkalns, E. V.; Scroggs. R E.; Mills, J. J. Med. Pharm. Chem. 1959, 1 , 223-243.
Table I.Solution-Phase [(M 2H)2+y[(M H)+] for Gramicidin S and 4,4'-Bipiperidinea measd pH in aqueous soln concn (mol/L)
gramicidin S
10-6
3 x 10-6 8x 10-5 3 10-5
6.80 6.57 6.27
10-4
1.6 10-4 3 10-4 8x
6.90 6.78 6.05 5.98 5.92
5.93
gramicidin S 4,4'-bipiperidine 2.3 x l o 3 3.9 x 103 7.8
io3
1.7
104
5.75 5.57
10-3
4 10-3 10-2
4,4'-bipiperidine
estimated soh-phase
l(M + 2H)*+I/l(M + H)+1
x
104
5.66
6.9 8.7
4.67
2.8 x lo3 3.3 103 3.8 x lo3 5.6 x 103
3.9
5.22
4.0 x lo2 5.2 x lo2
x
103
104
6.8 x lo4
pKal for diprotonated gramicidin S, f0.16. pKal for diprotonated 4,4'-bipiperidine, 9.5.
1 are the solution-phase calculated values of [MH22+1/[MH+las functions of concentration in aqueous solutions. It should be noted that the degree of protonation is augmented with increasing analyte concentration, as indicated by the increasing [MHz2+1/ [MH+] ratios for solutions of both analytes. When the solvent is changed from water to methanol, the pKa values of protonated nitrogen bases are expected to increase by approximately Analytical Chemistry, Vol. 67, No. 17, September 7, 7995
2895
1.2units,x” which will further increase the extent of protonation (Le., dissociation decreases in methanol). The increase in solution-phase protonation at higher analyte concentrations might therefore be expected to contribute to an increase in the average charge state observed in ES mass spech;l. Instead, just the opposite was observed, namely, lowering of charge states with increasing analyte concentrations in Figures 2b and 3b. Increasing the concentrations of analytes present in the salt form also leads to higher solution conductivities, resulting in augmented electrospray needle currents observed in ESMS experiments, hence, an increase in the total number of excess charges in “electrosprayed” droplets. To more closely examine the impact of this change on the observed anaiyte charge state distributions, we introduce the ratio,N/N,, the ratio of the total number of excess charges in all droplets 0to the total number of analyte molecules in all droplets (NJ. The total number of excess charges (N)is defined as the number of elemental charges in the droplets with no counterion. The number of excesscharges generated per second can be approximated hy the ES needle current indicating the rate at which positive charges leave the needle and arrive at the counterelectrode. The total number of analyte molecules (No)entering “electrosprayed” droplets per second can be deduced from the product of the &e eoncentm tion and the flow rate at which the solution containing analyte is fed into the electrospray needle. The following equation is used to calculate the N/No ratio:
where l i s the elecmspray current (Us), e is the elemental charge (1.602 x C/charge), A is Avogadro’s number (6.023 x 1023 molecules/mol). C i s the analyte concentration (mol/L), and II is the flow rate of the analytecontaining solution into the electrospray mass spectrometer Ws).The N/N, ratio is thus an indicator of the number of excess charges available per anaiyte molecule present, which has been plotted vs analyte concentration in Figures 2c and 3c. The process of droplet charging in positive ion ESMS may be the result of the electrochemical removal of anions at the metal capillary, in which case the excess positive charges contained in droplets arise from cations originally present in solution whose counterion has been removed. Alternatively, electrolytic oxidation of neutral species in solution or metal species on the ES capillary may be occurring to produce solution cations (eg., Ht, F@+, etc.).” It is likely that the actual scenario is a combination of both types of processes. In a situation where all excess charges in a droplet originate fromremoval of analyte eounterions (anions) and all excess charge ultimately is canied by analyte molecules, the N/No ratio would be a direct indicator of the efficiency of analyte charging in the electrospray process Furthermore, if all the excess charges in the droplet are represented by iniMly diprotonated analyte, the highest possible N/N. value is 2 in the case where all counterions have been removed. As the analyte concentration increases in bulk solution, a roughly proportional increase of the analyte species in the charged droplets is expected, while the number of excess charges only E. Z pkys. Gem. 1934.169A. 207-224. (34) (35) Ritchie. C. D.:Meperle, C. H.J Am. Cbm, Sor. 1967.89. 1447-1452. 2896 Analyiical Chemistry, Vd. 67,No. 17,September I. 1995
Rcsh.I”tm.l.
Gu-*Lm
b.dmIlmd.l(
Ou*m.
Figure 4. Schematic diagram of the outer portion of a charged
droplet containing an exms of positive charges. Doubly protonated analyte molecules (doubly charged ellipses)were initially introduced in the salt form with two anions (circles labeled A-) attached. Droplet charging wurred by electrophoretic removal of anions and electrochemical production of cations (smallest circles bearing i charges). The ellipses are arranged in a horizontal fashion for visual clarity. The anions may be considered to be located just below the nearsulface layer where excess positive charges reside. The middle panels show a later stage of the droplet lifetime where solvent has evaporated to decrease the droplet size. and the probability of forming contact ion pairs has increased. The diagram depicts the relation between Nl& (top)and the charge states of ES-generated gas-phase ions (bottom). Pathway b corresponds more closely to experimental observations
incream weakiy (I 0= , 0 . 2 < n < 0.4).% As a consequence, an increasing proportion of diprotonated analyte molecules located very close to the droplet surface have nearby counterions such as c1- (or other possible anions). The specific diprotonated analyte species that is closest to a particular anion and is thus receiving the bulk of the counterion iniluence can change rapidly from one diprotonated analyte species to another. To minimize
intramolecular Conlombic repulsion, available counterionswill be distrihuted widely over the total number of diprotonated species contributing to the charge excess near the droplet surface. We thus propme the presence of higher numbers of ion-paired analyte species to be largely responsible for reducing the proportion of doubly charged species without counterions in droplets at higher concentrations. As a consequence, a decreased number of doubly charged ga4phase ions are produced. To better illustrate the relation between the ratio @ 2H)”/ I @I H)+ observed in ES mass spectra and the N/N, ratio as measured by electrospray current and analyte concentration, Fire 4 and F i i r e 5 present schematic descriptions of an outer portion of a charged droplet containing dibasic analyte species at low and high concentrations, respectively. To preserve clarity in
+
(36)Tang. L; Kebarle. P.AWL Qla. 1991.63.2709-2715.
+
H+A- to yield two monoprotonated gasphase analyte molecules (ellipses bearing a single charge), which were originally absent in the droplet and three doubly charged analyte ions. Because the process represented hy path b, wherein available counterions attach to a larger number of multipiy protonated analyte molecules, leads to an analyte charge state distribution which more closely resembles that obselved in E 3 mass spectra, we propose path b to be chiefly responsible for the disparity between solution protonation equilibria and ESMS charge state distributions. It should be noted that available anions may also ion pair with other solution cations (smallest circles). F i r e 4 presents a situation where the atlinity of the anions for the elechochemically generated cations (smallest circles) is relatively high, resulting in the production of residual neutrals. If the initial concentration of analyte is raised such that the nnmher of analyte molecules in an otherwise identical droplet portion increases from 5 to 8 Figure 5), because of the weak dependence of the electrospray current I on the solution conductivity (Ia u",0.2 < n e 0.4),36 the number of excess charges increases only from about 8 to 10. In moving from the lower concentration situation Figure 4) to that of higher concentration Fire 5), the N/N, value would thus be lowered from 8/5 = 1.6 to 10/8 = 1.25, and there will he a higher number of Acounterions in the droplet Distribution of these counterions to stahilie the charge associated with as many diprotonated analyte ions as possible will ultimately result in a lowering of the overall charge associated with the analyte. This occurs because a higher proportion of protonated sites are charge neutralized by Acounterions via formation of contact ion pairs during droplet shrinkage F i r e 5, center panel). Singly charged analyte ions that are thereby formed (with one of the two protonated sites bearing an A- counterion) presumably desorb into the gas phase accompanied by the dissociation of a "neutral" H+A- molecule to form the mass spectmmehically observed MH+ (ellipse bearing single charge, Fire 5). The driving force for the departure of the HA molecule may be thermal, or it may be the result of collision-induced dissociation. At higher concentrations (Figure 5). the proportion of molecules undergoing this process is raised relative to the lower concentration case (Figure 4). hence, the average ESMS detected charge state (2) is reduced. Although these schematic diagrams depict small molecules bearing a maximum of two charges, the concepts brought forth are also applicable to rationalizing the shift in charge state distributions toward lower values for increasing concentrations of larger molecules, such as proteins. Additional evidence for the scenario described above is provided by the observation of OM 2H+ Cl-)+ species in ES mass spectra of both gramicidin S and 4,4bipiperidine obtained at a lower source block temperature (70 "C). Figure 6 shows singly charged ions originating from attachment of a CI- countenon to diprotonated 4,4'-BPD in absolute methanol, yielding (M 2H+ Cl-)+. Notably, cluster ions of the form (M 2H+ nHC02+ do not appear in ES mass spectra of either compound, suggesting that attached H+, CI- species should not be regarded as a "neutral" HCI molecule solvating the charge. Moreover, the intensities of (M 2H+ CI-)+ peaks increase as the concentrations are raised, which supports the proposed mechanism where a decreased N/N. ratio. or an increased proportion of ion-paired analyte relative to available excess charges, is responsible for the shifting of analyte charge state distributions to lower values.
+
I
Reddull r a n d s
G u p b kms
Figure 5. Schematic diagram of the outer pollion of a charged droplet containing a higher concentration of the same dibasic analyte, originally introduced in salt I o n , as shown in Figure 4. An explanation of symbols employed is given in Figure 4. The ObSeNed increase in the propollion of singly charged ES-generated ions at higher con-
centration is attributed largely to the reduced
" , value.
+
the diagrams, neutral nonanalyte species have been omitted. Monoprotonated and unprotonated analyte specieshave also been left out, which is justified by calculations showing that they are indeed negligible species in solution in the experimental pH range (see Table 1). Droplet charging is considered to occur by electrochemical oxidation of anions (products not shown) and by electrochemical oxidation of solution neutrals to generate positively charged species (represented by plus charges in the smallest circles). At the top of Figure 4, the outer droplet portion contains five doubly protonated analyte molecules (represented by ellipses with two plus charges). two electrochemically generated cations (the smallest circles bearing plus charges), and four anions (represented by circles labeled A-). With 12 positive charges and 4 negative charges, the droplet portion contains 8 excess positive charges. Thus the ratio of excess charges to analyte molecules, N/N,, in this representative droplet portion is 8/5 = 1.6. As solvent evaporation proceeds and the droplet reduces in size,ions of opposite charge are proposed to move closer together to form "contact ion pairs" F i r e 4, center panels). The formation of such contact ion pairs may involve attachment of two anions to one diprotonated analyte molecule, which will ultimately end up as a residual neutral along with four gasphase doubly charged analyte ions F i r e 4, path a). Alternatively, as depicted in path h, contact iorrpair formation may involve attachment of two anions to separate diprotonated molecules which subsequently dissociate
+
+
+
+
+
Analyiical Chemisty, Vol. 67, No. 17, September 1, 1995
2897
+
+
+
169 (M+H)' ,4000
3500
-P --
h
3000 2500
1
2000
-
1500
.
1000
1
.
.-
500
205 (M+tH++CI-)+
1 150
160
170
180
190
200
210
220
230
240
m/z Figure 6. Low-resolution positive ion electrospray mass spectrum of 4,4'-bipiperidine dihydrochloride acquired at lowered block temperature (70 "C)and minimal CID conditions.
It is important to point out that, in the highest concentration regimes in Figures 2c and 3c, the N/No values have diminished severely, while the analyte charge states (Figures 2b and 3b) decrease to a lower extent. In other words, the reduction in average charge state 2 observed in ES mass spectra is relatively minor compared to the major decrease in the total excess charges available per analyte molecule present in the initially formed droplets. One factor that could be contributing to this disproportionality in the rates of change of the average 2 and the N/No ratio with increasing concentration is the phenomenon of uneven fission Occurring in droplets. According to the recent findings of several a ~ t h o r s , during ~ ~ - ~ droplet ~ fission occurring at 70-80% of the charge-to-mass ratio corresponding to the Rayleigh instability limit, offspring droplets produced by fission carry about 15%of the charge and 2%of the mass of the parent droplet. This rule was obeyed until the droplets become too small to be observed. Thus, after one uneven fission event, the charge-tomass ratio in the offspring droplets may be enhanced by as much as 7-f0ld.~~*~j This implies that N/No for analyte species would also be increased to a considerable extent during such a process. Hence, a large initial decrease in NIN, due to a higher initial analyte concentration can be attenuated by a subsequent increase in NIN, in the offspring droplets generated in one or more uneven fission events. Another point that must be addressed is that at lower concentrations (e.g., < M) the N/Noratio increases well above 2 (Figures 2c and 3c), while the average charge state 2 is seen to asymptotically approach the maximum value of 2 = 2 (Figures 2b and 3b), corresponding to protonation of both nucleophilic sites on each analyte molecule. It was also observed that, at lower analyte concentrations, the electrospray current I leveled off at about (2.5-3.0) x lo-* A, which corresponded to the electrospray current for neat solvent (devoid of analyte). These combined observations suggest that at lower analyte concentrations where the N/No value surpasses a value of 2, it is nonanalyte species that determine the charge excess in droplets. Because solvent may contain up to 5 x M impurity electrolytes," this continued presence of significant background current at steadily decreasing trace analyte concentrations serves to maintain N at a substantial minimum level, while Noprogressively dwindles. This reasoning points to the electrolytic generation of charged species 2898
Analytical Chemistry, Vol. 67, No. 77, September 1, 1995
(e.g., H+, Fe2+) and/or the electrophoretic removal of OCH3- or impurity anions as the dominant contributors to the excess charges in droplets at low analyte concentrations. While not excluding possible contributions from gas-phase modifications of analyte charge states (e.g., collision-induced dissociations), these other sources of excess charges at low analyte concentrations may be contributing to the leveling off of analyte charge states below the upper limit value of 2. In other words, even at very low analyte concentrations, total removal of anionic counterions during charged droplet formation is apparently still not complete. In order to compare the effect of analyte concentration and that of external electrolyte concentration (i.e., ionic strength) on the charge state distributions of analytes, additional experiments were carried out in which CsCl was added in increasing amounts to solutions containing either gramicidin S or 4,4'-bipiperidine. Interestingly, the charge states observed in ES mass spectra of both gramicidin S and 4,4'-bipiperidine were perceptibly shifted to lower values with increasing concentrations of CsC1. These results contrast with the near constancy of charge state distributions observed upon addition of CsCl to higher molecular weight protein solutions shown in a previous publication.26 This indicates that in these small molecule systems CsCl is not a true "spectatoi' electrolyte (Le., it does participate in charge attachment/removal), and the effect of CsCl on charge state distributions thus appears to be analyte dependent. When protein solutions containing high concentrations of CsCl are subjected to the electrospray ionization process, if a protein molecule carries n charges in a droplet, attachment of C1- to this protein molecule (Le., neutralization of one H+) would leave one Cs+ without a counterion (constituting part of the charge excess) and a protein molecule with n - 1 charge: (protein)'+
+ CsCl - (protein + CI-)'"-"+ + CS+
Anion effects similar to the exchange of C1- described above have been observed by Mirza and Chait.37 Desorption of the protein molecule with n - 1 positive charges accompanied by desorption of Cs+ would thus cause a shift of the average charge states from 2 to 2 - 1, yet a shift of this magnitude was not observed.26 On the other hand, in the case of much smaller molecules such as gramicidin S and 4,4'-bipiperidine, increasing CsCl concentration caused an appreciable shift in charge state distributions toward lower values. In an analogous manner, "anion exchange" may take place as follows:
MH;+
+ c s c i - ( M H ~ ~++cr)++ CS+
The exact reasons why the charge state distributions for gramicidin S and bipiperidine are more variable as functions of CsCl concentration than those of proteins are not known. One possible explanation is that, given a common anion (Cl-), the desorption rate of positively charged analyte ions relative to that of Cs+ may contribute to determining the extent of anion exchange. In comparing the relative intensities of multiply charged protein peaks (Le., myoglobin or lysozyme) with Cs+ in a two-component equimolar mixture, the proteins exhibited much higher peak intensities than that of Cs+, indicating that the desorption rate constants for multiply charged protein ions are (37) Mirza, U. A: Chait, B. T. Anal. Chem. 1994,66, 2898-2904.
considerably larger than the desorption rate constant for Cs+. In other cases where ES mass spectra of equimolar mixtures of CsCl and 4,4'-bipiperidine or CsCl and gramicidin S were obtained, the intensities of 4,4'-bipiperidine and gramicidin S were only moderately higher than that of Cst, indicating a smaller difference between the desorption rate constants of the analytes and that of Cs+. In other words, a larger difference between the desorption rate constant of analyte ions and that of Cs+ may lead to a smaller degree of variability of analyte charge state with varying electrolyte concentration. According to Fenn's model,21at higher concentrations a larger number of analyte ions of lower charge state desorb during earlier stages of droplet evaporation, thereby depleting the excess charge available for desorption of higher charge state ions at later stages. For gramicidin S and 4,4'-bipiperidine, doubly protonated species dominate in solution phase. Generation of a higher proportion of singly charged gas-phase ions than were originally present in solution is likely to be accompanied by transfer of one proton from the doubly charged species to either a solvent molecule or to a counterion (Cl-) in solution. Solution-phase transfer of a proton to a water molecule, however, is highly endothermic (requiring a positive AG = 58 kJ/mol for gramicidin S and AG = 55 kJ/mol for 4,4'-bipiperidine) and thus may be considered as a negligible process. Proton transfer to methanol in solution is even less favored. It then seems that there is little possibility to desorb singly charged analyte ions in high abundances without prior transfer of one proton from the doubly charged forms to anionic counterions. Negative Ion ESMS. In separate negative ion experiments where Trypan Blue and eosin Y were used as analytes, the pH values of aqueous solutions of the two compounds were measured at different concentrations to determine whether resultant changes in [OH-] contribute to the variability of analyte charge state distributions in ES mass spectra. Solution-phase concentration ratios such as [(M - 4Na)4-1/[(M - 4Na (for Trypan Blue) and [ (M' - 2Na)?-]/[ ( M - 2Na H)-] (for eosin Y) can be estimated as follows:
+
+ H)"] (M' - 2 N a + H)-]
+ m3-]
[(M - 4Na)4-]/[(M - 4Na
= [OH-]/K,,
[ (M' - 2Na)'-]/[
= [OH-]/K,,'
where
are equilibrium constants for the reactions = (M - 4Na H)3- OH- and (M' 2Na)'- H20 (M' - 2Na H)- OH-, respectively. Aqueous solutions of both analytes showed slight increases in pH when their concentrations were raised, and the analyte charge level in solution should increase according to the above equations. This might be expected to result in the formation of a higher proportion of more highly charged analyte anions (higher 2) at elevated concentrations. However, similar to the positive ion results, desorption of a higher proportion of lower charge state analyte anions was observed at higher analyte concentrations in direct opposition to solution-phase predictions. Lower values of observed average charge states in the gas phase imply a higher degree of Na+ or proton attachment to analyte anions prior to gas-phase ion detection. As the analyte concentration increases, the ratio N / N , decreases due to the weak dependence of electrospray current on analyte concentration. As a result, analogous to the positive ion situation, at higher analyte concentrations droplets Kbl
and
&I'
(M - 4Na)4- + H20
+
+
+
+
+
a 4-
3. 2.
ilx'"'l 1 3
i
1-
1x10'
/
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
T'l
1x100
'1
2.5
c
b
-
+
-
1x102 C
1x10' O
Fz
1x100 1x10.' ~~
l
x iX10.'
1 0 . 1 ~ 1 0 . 6 1~10.s 1x10.4
2 iX10.3
I 1~10.~
Concentration (M) Figure 7. log-log plots of (a) abundances of singly charged and multiply charged negative ions, (b) average charge state Z, and (c) NIN, ratio, all vs concentration, for Trypan Blue.
contain more counterions, in this case, positive charge carriers such as Nat, H+, etc. The dependence of charge states of Trypan Blue on its solution concentration is shown in Figure 7. In general, as the concentration was raised, the absolute abundances of all analyte ions of different charge states first increased and then began to level off or even decrease as shown in Figure 7a. Notably, in moving in the direction of increasing concentration, the highest charge state ion (4-) began to decrease first, and ultimately decreased to the greatest extent. The 3- and 2- ions decreased later and to successively lower extents, while the 1- charge state ion never decreased. The resultant average charge state 2,plotted in Figure 7b,decreased from 3.5 to 2.6 over the range of concentration increase. It should be noted that, in calculating 2,protonated and natriated forms of a given charge state were directly summed. Also of note is that at concentrations below 3 x M (first two data points from left), the rate of change of average 2 values is less dramatic than at higher concentrations. The decrease of calculated N/No values with increasing analyte concentration, shown in Figure 7c, was taken as the underlying factor leading to the reduced 2 values at high concentrations. Despite the increased N/No at low concentrations, the observed 2 never rose above 3.5, even at the lowest concentration. Although the 2 values did approach the maximum value of 4 asymptotically, the fact that 3.5 was the highest average charge state obtained suggests that electrolytic generation of negatively Analytical Chemistiy, Vol. 67, No. 17, September 1, 1995
2899
3
1x10~ h
1x10'
a
-
21
b
5x1Oo-$
1x10-2
1
1 x 1 0 . ~ 1x10.5
1 x 1 0 . ~ 1x10.'
1x10.~ 1 ~ 1 0 . ~
Concentration (M) Figure 8. log-log plots of (a) abundances of singly charged and doubly charged negative ions, (b) average charge state Z,and (c) NIN, ratio, all vs concentration, for eosin Y.
charged species in combination with electrophoretic removal of impurity cations is largely responsible for the creation of excess negative droplet charge at low analyte concentrations. This factor results in an incomplete removal of cations (counterions) originally associated with anionic analyte, hence, the observation of lower gas-phase charge states than the maximum value (2 = 4). At higher concentrations, N / N , diminishes rather sharply with increasing concentration, which predicts a greater shift of charge states toward lower values than was observed. As discussed before for the positive ion mode, if uneven fission of droplets enhances the charge to mass ratio in negatively charged offspring droplets such that the actual NIN, ratio was raised in offspring droplets, then an augmented average charge state 2 would result in negative ion ES mass spectra. Also worth noting is that, at higher concentrations, Na+ attachment becomes increasingly dominant as compared to H+ attachment. This observation is consistent with the dominant role of Na+ as counterions in solution. In Figure 8a, the abundances of the doubly charged and singly charged eosin Y molecules displayed a dependence on concentration similar to the previous examples. From to M (first five data points from left), the singly charged ion abundance
2900 Analytical Chemistty, Vol. 67, No. 17, September 1 , 1995
increased monotonically while that of the doubly charged was rather constant up to 5 x M; above this level, the 2- ion abundance started to decrease as the concentration was further increased. Consequently, as shown in Figure 8b, the detected average charge state 2 diminished steadily with increasing concentration. Similar to the case of positive ion ESMS, the ratio of excess negative charge to the total number of analyte molecules (NIN,) was plotted as a function of solution concentration in Figure 8c. With increasing concentration of eosin Y, the NIN, value decreased more dramatically than the average 2 value, mirroring the positive ion results. CONCLUSION Analyte charge state distributions were found to decrease reproducibly with increasing analyte concentrations. These observations in both positive and negative ion ESMS for several selected compounds are consistent with previous reports from our laboratory14 and other research groups.6s"J* In positive ion ESMS, analytes employed in this work were introduced into solution in the protonated (chloride salt) form. Increasing concentrations of analyte species will increase the degree of protonation in solution phase, which contrasts with the lowering of analyte charge state distributions observed in positive ion ES mass spectra. This inconsistency was also observed in negative ion ESMS, where the degree of negative charge associated with analyte species in solution was increased with increasing concentrations, while the observed charge states were shifted toward lower values in the gas phase. In elucidating the mechanism responsible for the above observations, NIN,, the ratio of the number of excess charges (N) to the number of analyte molecules (No)in droplets, was introduced and its value was compared with the average analyte charge state value as a function of analyte concentration. It was found that NIN, decreased concurrently with analyte charge state as the analyte concentrations were increased. We propose that a decreasing N / N , value, which is manifested by an increasing number of ion pairs of multiply charged analyte species and counterions in the droplets, is chiefly responsible for yielding gas-phase ions of lowered charge states. Furthermore, the fact that the observed analyte charge states were not lowered as fast as predicted by the N / N , values was attributed to the uneven fission of droplets, which generates offspring droplets of enhanced charge to mass ratio. In this way, the value of NIN, for offspring droplets (from which gas-phase analyte ions originate) would actually be increased compared to the calculated N / N , values for initial droplets formed at the start of the electrospray process. ACKNOWLEDGMENT Financial support for this research was provided by the National Science Foundation and the Louisiana Stimulus for Excellence in Research. Received for review March 16, 1995. Accepted June 13,
1995.B AC950268M @
Abstract published in Advance ACS Abstracts, July 15, 1995.
