FROM IONS IN SOLUTION TO IONS IN THE GAS PHASE The Mechanism of Electrospray Mass Spectrometry
REPORT Paul Kebarle and Liang Tang Department of Chemistry University of Alberta Edmonton, Alberta Canada T6G 2G2
It is no exaggeration to say that close to half of inorganic chemistry, organic chemistry, and biochemistry involves ions in solution. A technique t h a t allows ions to be t r a n s f e r r e d from solution to the gas phase and s u b j e c t e d to m a s s s p e c t r o m e t r i c analysis should therefore be of enormous importance. Electrospray (ES) is such a technique. The transfer of ions from the gas phase to solution is a n a t u r a l process. In the presence of solvent molecules such as H 2 0 , naked gas-phase ions such as H 3 0 + or Na + will spontaneously form ion-solvent molecule c l u s t e r s such as H 3 0 + ( H 2 0 ) M a n d Na + (H 2 0)„. If the pressure of the solv e n t vapor is somewhat above t h e saturation vapor pressure, these clusters will grow to small droplets. To visualize this process one needs only to t h i n k of t h e Wilson cloud chamber, where such a process occurs in front of one's eyes. In the past 30 years mass spectro-
metric studies of ion-molecule clust e r s in the gas phase (1) have contributed much to the understanding of ion-solvent molecule interactions in solution. It is much easier to u n d e r s t a n d t h e n a t u r e of t h e i o n solvent bonding when the ion interacts with only one or a few solvent molecules, and it is these initial interactions t h a t dominate ion solvation in solution (I). The transfer of ions from solution to the gas phase is a desolvation process, and thus it is strongly "unnatur a l " or endoergic. The free energy r e q u i r e d w h e n a mole of N a + is transferred from aqueous solution to the gas phase is very large (2) Na + (aq) -> Na + (g) -AGml(Na+)
= 98.2kcal/mol
(1)
Analytical mass spectrometric methods in which ions are " t r a n s f e r r e d " from s o l u t i o n to t h e g a s phase, such as fast atom bombardment (FAB), plasma desorption, and laser desorption, existed before the i n t r o d u c t i o n of e l e c t r o s p r a y m a s s s p e c t r o m e t r y (ESMS). The e n e r g y required for ion transfer to the gas p h a s e in t h e s e e a r l i e r m e t h o d s is supplied by complex high-energy collision cascades and highly localized heating, resulting in additional processes such as net ionization (creation of ions from neutrals) and fragmentation of ions. ESMS, in contrast, is the
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"purest" form of transfer of ions from solution to the gas phase; little if any extra internal energy is imparted to the ions. ES affords ion transfer from solut i o n to t h e g a s p h a s e for a l a r g e range of ion types: the simple singly charged electrolytes such as Na + and Cl~ a n d organic p r o t o n a t e d b a s e s B H + and X"; group II ions such as Ca 2 + , Sr 2 + , and Ba , as well as doubly a n d t r i p l y c h a r g e d t r a n s i t i o n metal and lanthanide ions and complexes thereof; bioorganic ions such as multiply-protonated peptides and proteins of molecular m a s s 100,000 Da; and multiply-deprotonated, negatively charged nucleic acids. ESMS was introduced by Yamashita and Fenn in 1984 (3), and it took a few y e a r s before the importance of the method was recognized (4-6). Rec e n t a d v a n c e s (7-9) c o n f i r m t h e widely held view t h a t E S h a s u s h ered in a new era of the mass spectrometric analysis of biomolecules. The exciting applications of ESMS have also created g r e a t i n t e r e s t in t h e m e c h a n i s m by which t h e g a s p h a s e i o n s r e q u i r e d for t h e m a s s spectrometric analysis are produced by the ES device. Ideally, an unders t a n d i n g of t h e m e c h a n i s m should not only provide aesthetic satisfaction, it should be of significant practical use. Understanding the mechanism essentially provides a m e n t a l m a p t h a t allows planning of experi0003-2700/93/0365-972A/$04.00/0 © 1993 American Chemical Society
m e n t s and making rational choices of experimental parameters leading to a desired result. Unfortunately, the understanding of the ES mecha nism is not yet complete. Neverthe l e s s , t h e w h y s a n d w h y n o t s for many experimental p a r a m e t e r s are understood. What is still uncertain is exactly how the g a s - p h a s e ions are produced from t h e very small a n d highly charged droplets. Readers of this REPORT will learn about current i d e a s b u t will not find c e r t a i n t y . However, much useful and interest ing information is obtained about the conditions existing in ESMS when one attempts to pin down the mecha nism involved. Fortunately, for prac tical purposes, the predictions of the two presently favored m e c h a n i s m s turn out to be quite similar. ESMS has a great advantage over the other methods because the ionic solutions used for transfer of ions to the gas phase are the same solutions used in conventional wet chemistry. No u n u s u a l liquid matrices such as the glycerol used in FAB and matrixassisted laser desorption ionization are required. Now t h a t good ESMS interfaces are available on commer cial m a s s spectrometers, the u s e r s and not the interface developers are the important players. These u s e r s a r e t y p i c a l l y w o r k e r s in s o l u t i o n chemistry. Once t h e s e i n d i v i d u a l s have acquired some u n d e r s t a n d i n g of the mechanism, they will be in a
position to use the full range of their background and imagination to de velop new applications of the ESMS technique. ES existed long before its applica tion to MS. It is a method of consid erable i m p o r t a n c e for t h e electro s t a t i c d i s p e r s i o n of l i q u i d s . T h e i n t e r e s t i n g history and notable r e search advances are very well de scribed in Bailey's book, Electrostatic Spraying of Liquids (9). This research also provides the basis for the ESMS mechanistic studies. The new aspect in the m a s s spectrometric applica tion concerns the production of gasphase ions from the ions in the solu tion; this was of no i n t e r e s t to the spray researchers (9). The ES mechanism: Features of importance to ESMS T h e r e a r e four major processes in E S M S : t h e p r o d u c t i o n of c h a r g e d droplets from electrolyte dissolved in a solvent; shrinkage of charged drop lets by solvent evaporation and r e peated droplet disintegrations (fis sions), l e a d i n g u l t i m a t e l y to very small, highly charged droplets capa ble of producing gas-phase ions; the mechanism of gas-phase ion produc tion; a n d s e c o n d a r y processes, by which gas-phase ions are modified in the a t m o s p h e r i c a n d t h e ion s a m pling regions of the spectrometer. Al though this last topic is important, it includes a wide area of phenomena,
some of which are not well u n d e r stood. Therefore these effects will not be considered here. Production of charged droplets at the ES capillary tip As shown in the schematic represen tation of the ES events in air at at m o s p h e r i c p r e s s u r e ( F i g u r e 1), a voltage Vc of 2 - 3 kV is applied to the m e t a l capillary, which is typically 0.2 mm o.d. and 0.1 mm i.d. and lo cated 1-3 cm from the large planar counter electrode. In ESMS this counter electrode has an orifice lead ing to the mass spectrometric sam pling system. Because the capillary tip is very narrow, the electric field Ε in the air at the capillary tip is very high (E » 10 e V/m). When the capillary of radius rc is located at a distance d from the pla nar counter electrode, the magnitude of Ec for a given potential Vc is given by (10, 11) E=2V/rln(4d/r)
(2)
Equation 2 provides the field a t the capillary tip in the absence of solu tion. The field Ec is proportional to Vc, and the most important geometry parameter is rc. Ec is essentially in v e r s e l y p r o p o r t i o n a l to r c ; it d e creases slowly, with a logarithmic dependence, with the distance d. For e x a m p l e , w h e n Vc = 2000 V, rc = 0.2 m m , a n d d = 2 cm, Ec = 3.3 χ 10 6 V/m.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 22, NOVEMBER 15, 1993 · 973 A
REPORT The typical solution used in ESMS consists of a dipolar solvent in which electrolytes are a t least somewhat soluble. We use methanol as the sol vent and NaCl or BHC1 (where Β is an organic nitrogen base) as the elec trolytes. Essentially, these electro lytes are totally dissociated into Na + and Cl~ as well as BH + and Cl~ in the solvent. Low electrolyte concen trations, routinely in the 10" 5 -10~ 3 M range, are required for the opera tion of ES. The imposed field Ε will also par tially penetrate the liquid at the cap illary tip. To simplify the discussion, we will assume that all experiments are c a r r i e d out in the positive ion mode, which has a positive capillary potential. When the capillary is the positive electrode, some positive ions in the liquid will drift toward the liq uid surface and some negative ions drift away from it until the imposed field inside the liquid is essentially removed by t h i s charge redistribu tion. However, the accumulated pos itive charge a t the surface leads to déstabilisation of the surface because t h e positive ions are d r a w n downfield but cannot escape from the liquid. The surface is drawn out downfield such t h a t a liquid cone forms. This is called a Taylor cone, after the researcher who was one of the first to investigate the conditions u n d e r which a stable liquid cone can exist with the competing forces of an electric field and the surface tension of the liquid (12). At a sufficiently high field E, the cone is not stable and a liquid filam e n t with a d i a m e t e r of a few micrometers, whose surface is enriched on positive ions, is emitted from the Taylor cone t i p . At some d i s t a n c e downstream, the liquid filament becomes unstable and forms separate droplets. The droplets' surfaces are enriched with positive ions for which there are no negative counterions in t h e droplet (i.e., t h e d r o p l e t s a r e charged with a n excess of positive electrolyte ions). The length of the unbroken liquid filament decreases if t h e field Ε is increased. At h i g h e r fields, a m u l t i s p r a y c o n d i t i o n is r e a c h e d in which the c e n t r a l cone disappears and droplet emission oc curs from a crown of four to six short liquid tips formed at the rim of the capillary (13). We came to the conclusion that the ion separation described above is the mechanism responsible for droplet c h a r g i n g , l a r g e l y on t h e b a s i s of mass spectra observed with electro lytes in the K T 5 - l < r 3 M range (13, 14). The positive and negative ions
Reduction
Oxidation Etectrons
Electrons High-voltage power supply
Figure 1. Schematic representation of processes in ESMS. The very high electric field imposed by the power supply causes an enrichment of positive electrolyte ions at the meniscus of the solution at the metal capillary tip. This net charge is pulled downfield, expanding the meniscus into a cone that emits a fine mist of positively charged droplets. Solvent evaporation reduces the volume of the droplets at constant charge, leading to fission of the droplets. Charge balance is attained in the ES device by electrochemical oxidation at the positive electrode and reduction at the negative electrode.
observed in the spectra were always the positive and negative ions of the electrolytes present in the solution. Extraneous ions were observed only at high capillary voltages where elec tric (corona) discharges were occur ring at the capillary (15, 16). T h e ion s e p a r a t i o n m e c h a n i s m , which is called t h e electrophoretic mechanism, is also most plausible on energetic grounds. Electrical double l a y e r s a r e a l r e a d y formed a t low fields in electrolyte solutions. The resulting p o s i t i v e - n e g a t i v e ion r e d i s t r i b u t i o n reduces or completely removes the imposed field and there fore suppresses other forms of ion ization such as ionization by electron removal from molecules (field ioniza tion), which requires very high elec tric fields. Another way to show t h a t ES r e sults from electrophoretic charging is to deionize the solvent. Experiments involving methanol deionized by dis tillation, which reduced the conduc tivity from 1 0 ~ e n _ 1 c m * ( r e a g e n t g r a d e m e t h a n o l , ~ 1 0 ~ 5 M in i m p u r i t y e l e c t r o l y t e ) to 10~ 7 Ω~ 1 c m - 1 , showed t h a t t h e E S c u r r e n t decreased and became intermittent. The intensity of the observed impu rity ions (NH4, N a + ) present in re agent-grade methanol became much
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lower in the purified solvent, and the signal fluctuated widely on a t i m e scale of seconds (see Figure 3 in Ref erence 14). If the charge separation is electro phoretic, at a steady state the posi tive droplet emission will continu o u s l y c a r r y off p o s i t i v e i o n s . C o n s i d e r i n g t h e r e q u i r e m e n t s for charge balance in such a continuous electric current device, and that only electrons can flow through the metal wire supplying the electric potential to t h e e l e c t r o d e s ( F i g u r e 1), one comes to the conclusion t h a t the ES process should involve a n electro chemical conversion of ions to elec trons. In other words, the ES device can be viewed as a special type of electrolytic cell in which that part of t h e ion t r a n s p o r t does n o t occur through uninterrupted solution, but as charged droplets and later as ions in the gas phase. Therefore, a con ventional electrochemical oxidation reaction should be occurring a t the l i q u i d - m e t a l interface of the capil lary. This reaction should be supply ing positive ions to the solution by converting metal atoms to positive ions and electrons or by converting negative ions from t h e solution to n e u t r a l molecules a n d a n electron. This conversion process is accom-
Table 1. Onset voltages Von and surface tension γ for ES with different solvents
V„(M)
Y(N/m2)
CH3OH
CH3CN
(CH3)2SO
H20
2.2
2.5 0.030
3.0 0.043
4.0 0.073
0.0226
pushed by oxidation reactions such as M (s) -> M2+(aq) + 2e" 4 O H l a q ) -> 0 2 (g) + 2 H 2 0 + 4e" (3)
onset of c h a r g e d - d r o p l e t emission (i.e., t h e o n s e t of E S ) . W h e n h i s equation is combined with Equation 2, which relates the field Ec to the potential V, one obtains
assuming t h a t an aqueous solution is electrosprayed. The actual lowest ox idation potential reaction expected to occur will depend entirely on the sol vent and composition of the solution used. Proof for the occurrence of elec trochemical oxidation a t t h e metal capillary was provided by Blades et al. (17). When a Zn capillary tip was used, the release of Zn 2+ to the solu tion could be detected with ESMS. Furthermore, the amount of Zn 2 + re leased to the solution corresponded to the amount required to carry the ES current. Similar results were ob served with stainless steel capillaries that release Fe 2 + to the solution. The ions added to the solution by the electrolytic process are at a very low concentration, - 2 χ 10" 6 M (17). T h e y can be d e t e c t e d by MS, b u t t h e y do not, in g e n e r a l , i n t e r f e r e with the detection of other ionic analytes p r e s e n t in t h e solution. The work reported by Van Berkel et al. (18) shows some very interesting re sults when dry nonprotic solvents are used. There is little explicit discussion in the pre-MS ES literature concerning the nature of the charge carriers in the droplets. Although this is a vital question to the mass spectrometrist, it is of limited interest in other appli cations of ES. Pfeifer and Hendricks (11) were probably the first authors who explicitly p r o p o s e d a n d d i s cussed t h e electrophoretic m e c h a nism. More recently, Hayati, Bailey, and Tadros (19) also explicitly en dorsed the electrophoretic charging mechanism in a comprehensive ex amination of features of the ES pro cess. However, neither research group considered the electrochemical n a t u r e of the process a t the m e t a l liquid interface. In a treatment of the cone instabil ity a n d c h a r g e d - d r o p l e t emission, S m i t h (20), who also a s s u m e d t h e electrophoretic mechanism of charg ing, provided a very useful equation for the electric field required for the
Von - 2 χ 10V c ) y 2 ln(4
