Mass and Charge Distribution Analysis in Negative Electrosprays of

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Mass and Charge Distribution Analysis in Negative Electrosprays of Large Polyethylene Glycol Chains by Ion Mobility Mass Spectrometry Ernesto Criado-Hidalgo,†,‡ Juan Fernández-García,† and Juan Fernández de la Mora*,† †

Yale University, Mechanical Engineering Department, New Haven, Connecticut 06520, United States SEADM, Boecillo, Spain

‡

S Supporting Information *

ABSTRACT: The mass spectrometric (MS) complexity associated with the quasi-continuous distribution of mass and charge (m, z) of electrosprayed industrial polymers may be moderated by use of ion mobility spectrometry (IMS) and MS in series. However, when the high charge levels typical of polar polymers stretch the gas phase ions into linear configurations, the mobility Z tends to be closely correlated with m/z, and IMS-MS does not yield spectra more readily interpretable than pure MS spectra. Here we note that the usual high charge states observed in the ESI of polyethylene glycol (PEG) arise because the stretched gas phase chain is able to strongly bind solution cations. We weaken this binding and therefore moderate the charge level by electrospraying in negative mode (NESI). This produces exclusively globular gas phase ions. IMS-MS then readily separates into distinct bands the different z-states, enabling an unambiguous assignment of all ions and simplifying the determination of mass distributions fz(m) for each charge state. The measured probability pz(m) that a polymer ion of given mass m will carry z charges spans a surprisingly narrow z range, each mass being present at most in two charge states. PEG ions of a given charge state z become unstable at a critical mass, below which they shed just one elementary charge, evidently by ion evaporation. We argue that NESI-IMS-MS offers significant analytical advantages over alternative methods previously demonstrated, particularly at increasing masses, when individual ion peaks can no longer be discerned.

D

extreme levels of charge reduction preserving the many advantages of mass spectrometry over pure IMS. There are presently a number of unknowns obstructing the development of IMS-MS analysis of polymers under moderate charge states. Among them are the need to (i) understand and control the charge states at which the polymer ions form naturally in ESI, (ii) understand the shapes acquired by these ions in order to make quantitative use of the mobility variable measured in IMSMS analysis. Point (ii) has been previously broached.12,13 Point (i) is our focus here and has been previously addressed through a number of strategies, such as the use of amines as chargereducing reagents in the electrospray region or the ES buffer,14,15 or the implementation of post-ES charge-reducing approaches.16,9,17 Post-ES charge-reduction has shown considerable promise,18 but requires specialized instrumentation, and comes often at the cost of a reduced sensitivity or an increased time of analysis. Accordingly, it would be preferable to achieve the ideal level of charge via optimal selection of the electrospraying conditions. An important step in this direction is the observation that gas phase addition of diethylamine, triethylamine,15 or of the superbase 1,8-diazabicyclo[5.4.0]-

etermining mass distributions of industrial polymers by electrospray ionization mass spectrometry (ESI-MS) is complicated by the high spectral congestion associated with the coexistence of multiple charges and masses.1 Ion mobility separation coupled to mass spectrometry (IMS-MS) offers better chances of overcoming this complexity than MS alone.2−7 However, even this approach fails in the common case of polymers that adopt stretched, relatively linear gas phase configurations, as a result of the high charge imparted by the electrospray process. The problem is that the electrical mobility Z of a linear ion is approximately proportional to its charge and inversely proportional to its length, hence Z is linear with z/m.8 Mobility is therefore highly correlated with m/z, so that resolving ions in an IMS-MS spectrum is almost as hopeless as in a pure mass spectrum, particularly at polymer masses of tens of thousands of Daltons. A reasonable strategy for successful IMS-MS analysis of polymers would be to achieve moderately low charge states, such that most ions would shift from primarily linear to predominantly globular structures. An extreme form of this method already used successfully is based on reducing z to unity.9−11 In this case, the mass spectrometer is severely restricted by its modest m/z range. However, since z = 1 and the ions are all globular, mobility analysis alone suffices to describe the mass distribution. Here we are concerned with less © 2013 American Chemical Society

Received: October 27, 2012 Accepted: January 14, 2013 Published: January 14, 2013 2710

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undec-7-ene (DBU)14 to the ES-source chamber produces charge states associated mostly with globular structures. The charge level is in fact so modest that pure mass spectrometry produces well resolved spectra from which mass distributions of polyethylene glycol (PEG) have been obtained with commercial deconvolution algorithms, at least in the case of chromatographically purified polymer samples of very narrow mass distributions.15 Our group has explored various buffers based on formates of organic amines (including dimethylammonium formate and triethylammonium formate).12,13 We have achieved conditions in which a small but measurable fraction of PEG ions adopt globular or transitional gas phase structures, providing considerable detail on the charge and mass dependence of the shapes of large PEG ions.12 However, most of the ions reaching the IMS are still in difficult to resolve fully stretched configurations. It is possible to eliminate essentially all nonglobular geometries by activation of the ions as they enter the vacuum system of the MS,14,15,13 but this comes at a cost. In our IMS-MS instrument, where the mobility is measured at atmospheric pressure, some of these ions lose charge upon entering the MS analyzer, therefore having different charge states in the mobility and the mass measurements. This then introduces an additional level of complexity in the process of inferring mass distributions from two-dimensional IMS-MS spectra.12,13 Substantial charge loss evidently takes place upon entry into the MS in the work of Bagal et al.14 and Huang et al.,15 although it is not recognizable either because no IMS measurement was taken or because it took place following the energetic entry of the ions in the MS. The cost in this case is not a complex IMS-MS spectrum (in fact, even the pure MS spectrum may be fairly simple15) but the difficulty to distinguish between the rather different mechanisms controlling the charge state in either the electrospray source or the vacuum interface. Much therefore remains to be understood on the rules determining the charge state of electrosprayed polymer ions and on its control for successful mass distribution analysis. For this reason, we will first review the basic information available on polymer charging and from it formulate a hypothesis that leads to a successful electrospraying strategy to achieve globular gas phase PEG configurations in the ion source region at atmospheric pressure. The results obtained will finally be exploited to sharpen the initial hypothesis on the ideal charging mechanism.

charge as they can possibly hold, has received some additional attention.20,21 These interesting details will hopefully be clarified in future studies. For our present purposes, however, it is enough to note that one possible strategy to limit the charge on the gas phase polymer ion is to identify solution ions that will bind considerably more weakly (or not at all) to the polar groups in the chain. That this strategy can be successful has become recently clear in a study of polymers such as polystyrene (Psty), where the level of charging is vastly smaller than in PEG, resulting always in globular gas phase ions.22 Figure SI1 (Supporting Information) shows how a sample of Psty with a narrow mass distribution centered at about 9 kDa produced by positive ESI is primarily singly charged, with a small proportion of doubly charged ions. In contrast, a typical PEG ion of 9 kDa takes up to about 18 charges. The relevant point here is that the absence of polar groups in the Psty backbone limits the attachment of solution ions to it. It follows that an effective means to avoid stretched polymer ions (i.e., to promote globules) is to rely on buffer ions that do not bind strongly to the polymer chain. Given that even the largest cations previously used to electrospray PEG solutions have formed primarily stretched ions under atmospheric conditions,12 the more successful approach used in the present study is to electrospray in the negative mode, relying on the poor affinity between anions and the polar ether groups in PEG.

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EXPERIMENTAL SECTION True mobility and m/z separation was achieved with a parallel plate differential mobility analyzer (DMA; SEADM model P4, resolution ∼50) in series with a quadrupole-TOF mass spectrometer (MDS Sciex model QStar XL; m/z up to 40 kDa), as previously described,23,24 and as sketched in Figure 1.

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CHARGING MECHANISM The pioneering ESI studies of Fenn and colleagues of PEG first described the formidable problem of spectral complexity.1 They also computed the maximal charge that could bind to the polar groups in the fully stretched PEG chain against Coulombic repulsion, obtaining fair agreement with the singularly high charge states z observed. This left little room for doubt on the highly elongated form of these ions and on the substantial binding energy between the polymer and the individually attached cations (Na+ in their work). Mobility measurements later confirmed the linear structure of highly charged PEG ions, showing also that lesser charge led to more compact shapes, including globular geometries below a critical charge.8 These earlier inferences have been refined by subsequent work on other polymers,19 where modeling and mobility measurements show that the elongated chain binds to the cations by wrapping 8−12 monomer linkers around them in a beads-on-a-string configuration resembling a rosary.5,13 The issue of how such ions are first formed from the solution, carrying almost as much

Figure 1. Schematic of the experimental setup.

Polymer solutions (∼10−100 μM) in MeOH−water 75:25 (vol.) containing typically 30 mM dimethylamonium formate (Met2AF) were electrosprayed in the negative polarity from the tip of a silica capillary with a 20 μm inner diameter and an outer diameter of 360 μm, tapered down to approximately 40 μm. Samples were driven through the capillary with backing pressures of up to 500 mbar. The electrospray source was operated in the cone-jet mode by raising it to some −2 kV with respect to the upper electrode of the DMA. In order to limit 2711

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Figure 2. Analysis of a relatively broad sample of PEG 14k. (a) IMS-MS spectrum (391 different voltages at 10 V intervals, 2 s per mass spectrum, total accumulation time: 13.02 min). (b) Low resolution (Δm = 44 Da) charge-resolved abundance distributions Cz(mx). (c) Mass distribution (inset at full resolution). (d) Low resolution charge distribution pz(mx) as defined in eq 3

used as calibrant, whose mobility in CO2 has been reported by Ude.25

charge loss in the atmosphere−vacuum transition region, the voltages of all electrostatic lenses in the region between the critical orifice and the skimmer (i.e., the declustering and focusing potentials at the mass spectrometer interface) were set down to 0 volts. The success of this approach will be later confirmed by the fact that most polymer ions transmitted by the DMA (operating under thermal conditions) preserve their original charge acquired at atmospheric pressure throughout the MS. The exceptional ions losing one charge at the MS interface are easily identified (and charge-corrected) because they occupy unnatural positions in the DMA-MS plane. The charge distributions obtained are therefore highly representative of what is originally produced by the electrospray source. Charged polymer particles are drawn electrostatically against a counterflow from the capillary tip into the DMA, whose recirculating gas sheath flow was CO2 at 22 °C. All measured mobilities therefore are in CO2 gas. The resulting monomobile ion beam is then mass-analyzed in the mass spectrometer without mass-selection or fragmentation. Scanning the voltage in the DMA produces a mass spectrum at every mobility and results in a set of mobility-selected mass spectra (the twodimensional IMS-MS spectrum), which we store as a single file. Linear conversion of DMA voltage into inverse ion mobility was obtained with tetraheptylammonium+ in the positive mode

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RESULTS

Figure 2a shows an IMS-MS spectrum of PEG 14k sprayed in the negative mode in methanol−water (75:25 vol.) 30 mM in Met2AF. Ion abundance is given in a logarithmic color scale versus DMA voltage and m/z. The various charge states z are indicated as −z in white letters on the right of the figure. This is the first reported spectrum for electrosprayed PEG where all the ions naturally produced at atmospheric pressure are globular, as evident from the lack of sharp turns (resulting from shape transitions) in the various blue-yellow-red bands seen, each associated with variable masses m and the z indicated in the figure. We shall refer to these bands as z-bands. The assignment of z is straightforward. The bands denoted z = 1, z = 2, and z = 3 show well resolved mass peaks and are readily identified by the spacing of 44/z Da between neighboring peaks within the band. This direct procedure fails at higher z and n, as the peaks become more blurred. However, because the bands are well separated from each other, their corresponding z is simply given by their order. Additional confirmation of both the correct z assignment made and the globular shape is included in the Supporting Information, based on converting m and the 2712

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collision cross section (Ω ∼ z/Z) into the corresponding mass and mobility diameters of a sphere, dm, d. For this task we follow earlier work,26 while also correcting for the attraction between the ion and the dipole it induces in the gas molecules. This collapses almost exactly most data into a single line (Figure SI2 in the Supporting Information), the exceptions being all associated with unstable ions having lost one charge on their way to the vacuum system of the MS. This anomaly is removed once the m/z value from the MS is correctly based on the original z value at the DMA (zDMA = zMS + 1). Similar unstable ions appearing in Figure 2a are marked by a white arrow joining doubly charged precursors to singly charged product ions. This instability is a confounding factor when determining mass distributions, but it may be identified in the original 2D spectrum (and avoided in the inversion procedure) because the ions having undergone charge loss do not ordinarily fall within a conventional z-band. Clustering. Among the numerous high signal intensity regions (red) seen in Figure 2a, only the one with z = 2 is associated with a single chain. The other high intensity regions correspond to aggregates of several chains of PEG 14 k (dimer, trimer, ...), resulting from the high solution concentrations used (100 μM). Distinction between the various aggregates can be clearly made by eye in similar data obtained for samples of narrower mass distribution (Figure SI3b in the Supporting Information for PEG 12.6k and Figure SI4 in the Supporting Information for PEG 6k), where charge loss transitions are also more visually obvious. The practical advantages of using large PEG concentrations are that we span with a single sample a wide mass range via clustering, we gather in a single spectrum a large amount of information, and (thanks to the increased signal) we can afford faster acquisition times. Spectra more representative of the dissolved mass distribution, obtained at concentrations down to 10 μM, are reported in Figure SI5 in the Supporting Information for PEG 6 kDa, 12.6 kDa, and 20 kDa. Mass Distributions. The segregation of the various charge states into well resolved z-bands makes the conversion of the IMS-MS spectrum of Figure 2a into a polymer mass distribution relatively straightforward. The m/z variable for each z-band is turned into an m variable through multiplication by the corresponding z. In order to obtain the z-resolved mass distribution Cz(m) shown in Figure 2b, we first create imaginary inclined lines in Figure 2a halfway between the centers of each z-band, thus deciding which ion counts are assigned to one value of z or the other. The precise position of these imaginary borders has little effect on the results because, as seen in Figure 2a, the various z-bands are well separated, even at z values as large as 9. The value of Cz(m) within a zband is obtained by summing all the corresponding ion counts contained in a mass interval (m, not m/z) of 44 Da centered around the discrete set of masses mx = mend + xm1 + zmi

(x integer)

multiplied by the aggregation level n) and for PEG ions attached to anions other than formate. No discretization is required in principle, since we measure m/z with a resolution of 10 000 and know z exactly. For example, the small piece of the mass distribution shown in the inset of Figure 2c is obtained at full resolution. The full range low-resolution mass distribution f(mx) shown in the same figure is obtained by summing the Cz(mx) data over all z: ∞

f (mx ) =

∑ Cz(mx) z=1

(2)

where no correction for the dependence of the ion transmission through the DMA and the MS or the detector counting efficiency has been implemented. Figure 2c shows clearly a series of peaks with mean masses centered at multiples of the monomer mass, extending up to at least 150 000 Da. Although we have not yet explored the upper mass limit achievable, it is clear that negative electrospray ionization-IMS-MS solves the problem of mass distribution determination up to unusually high masses. The low charge states used call for a mass spectrometer with a substantial m/z range [(m/z)max = 15 000 Da in Figure 2]. This range is not very common in ESI-MS systems, but it is commercially available, has been exploited in earlier polymer studies,13−15 and is drastically less demanding than the m/z range required by MALDI.27,28 Charge Distribution Probability. On electrospraying a PEG chain of mass m, it will form ions carrying a fixed number z of charges with a certain probability pz(m), which we determine by normalizing the Cz(mx) curves with the mass distribution f(mx) defined in eq 2: pz (mx ) = Cz(mx ) f (mx )

(3)

pz(m) distributions for PEG 14k are shown in Figure 2d. The range of charges spanned by a given m is surprisingly narrow, each mass being present at most in two charge states. It is noteworthy that these pz(m) curves show smooth single peaks (as expected on physical grounds) in spite of the convoluted form of the underlying Cz(m) curves, each of which displays a diversity of singular features such as maxima, minima, and inflections. These features are inevitable in view of the spiky nature of the mass distributions (Figure 2c), but they would survive in the derived pz(m) curves if the assumption made of a relatively constant ion counting and transmission efficiency were not approximately true. The success of this crude assumption is evidently due to the relatively narrow range of masses spanned by each z. The flat high-mass end of the p9(m) distribution included in Figure 2d is not realistic due to the incomplete description of the C10(m) distribution. The rising side of the p2(z) curve is barely captured by the data of Figure 2d, as the corresponding low masses are on the very edge of this sample. This region has been studied with a lower molecular weight sample (the same PEG 6k used for Figure SI2 in the Supporting Information), with results shown in Figure SI4 in the Supporting Information. Mechanism Controlling the Charge State. The simplicity of the charging probability curves in Figure 2d and Figure SI4 in the Supporting Information and, in particular, the relatively narrow range of PEG masses capable of acquiring any given number z of charges, is unusual. The familiar distribution of charges associated with electrosprayed protein ions is drastically different, even for proteins electrosprayed under native conditions.29 Typical ESI-MS spectra of PEG in positive

(1)

where m1 =44 Da is the mass of the repeating CH2−O−CH2 linker unit, mend = 18 Da is the combined mass of the two end groups (OH + H), the integer x is the number of individual linkers in the ion, z is the charge state, and mi the mass of the charger ions. Our choice of mi = 45 Da corresponding to the formate anion is unsure because no accurate high mass calibration was performed. The discretization (1) must be understood as a convenient device for fast data manipulation. It is evidently incorrect for PEG aggregates (where mend must be 2713

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range of charge states studied here but also that the gap between mmin and m* increases even further at larger z. Data on mmin(z) and m*(z) are also collected in Table SI1 in the Supporting Information. Figure 3 includes mmin(z) values obtained from a diversity of samples, showing a certain scatter due to the difficulty of determining precisely the minimum mass present at each charge state. This determination is based on the z-selected mass distributions shown in Figure 2b and is subject to some ambiguity due to the presence of occasional tails in some of the distributions. These variations are nonetheless modest at the scale of the much larger differences between the smallest masses produced by our electrospray and the stability limit m*(z). We therefore conclude that our buffer will produce globular PEG ions at masses far larger than the ∼150 kDa investigated here.

mode or of a protein in denaturing solution show a very large number of charge states for a given mass, while a given polymer mass here produces two charge states on the half-height region of the distributions and primarily one in the center of the distributions. Particularly noteworthy is the fact that the left tail of the mass distribution for z ions decays very much as the right tail of the z − 1 ions rises (in other words, the sum of the two is close to unity). This must be the result of a kinetic mechanism by which the z ions are converted into z − 1 ions, evidently via loss of a single charge. The process determining the charge level on these PEG ions is therefore single ion evaporation. In fact, the distributions pz(mx) observed here are almost identical to those previously observed for ionic liquid nanodrops for which the charge state was undoubtedly governed by ion evaporation.24 This mechanism makes sense in a situation where the polymer is unable to bind charge to a chain site, so it remains in solution. At the end of the evaporation process, the surface of the polymer globule is left with the relatively high charge previously held by the drop. This initial level of charge is far more than the polymer surface can retain, so much of it evaporates rapidly until a sustainable z value is achieved. The distributions p1(mx) of singly charged ions are expected to extend to fairly low masses due to the lack of Coulombic repulsion. In a PEG 2k sample we observe OH-PEGx-Hformate− ions with x as small as 11 (section SI.8 in the Supporting Information) Minimum Mass Carrying a Given Charge. We have shown (see also SI.1 in the Supporting Information) that all our negatively charged PEG ions are globular and therefore readily analyzable via IMS-MS. We now proceed to show that this favorable situation will continue at PEG masses beyond those studied here. The experimentally observed critical mass below which PEG globules lose stability at a Rayleigh-like limit is given approximately by m*(z) ∼ 500z2 for z values up to 5,8 and m*(z) ∼ 635z2 for larger z.13 Globular clusters are therefore guaranteed as long as the smallest observed electrosprayed PEG mass carrying z charges, to be denoted mmin(z), is larger than m*(z). These two special masses are compared to each other in Figure 3, showing that the desired condition mmin(z) > m*(z) is not only well verified within the

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DISCUSSION The IMS-MS approach used has given fundamental new information on the charge state of PEG ions as electrosprayed, which would have been difficult to obtain by alternative means. This has provided for the first time a quantitative picture of the charging probability distributions, pz(m), and the physical mechanism controlling ESI charge levels in the absence of strong binding between the chain and the charging ions. In addition to this, we now propose to compare the practical analytical potential of our negative electrospray ionization-IMSMS method with previous successful approaches for MS analysis of PEG or other similarly polar polymers. Huang et al.15 have analyzed chromatographically purified PEG 40 kDa of exceptionally narrow mass distribution (polydispersity of 1.001), extracting impressive deconvoluted mass distributions from pure TOF-MS data. However, their data include essentially no overlapping between charge states, while a substantial level of overlapping would be hard to avoid in broader distributions, with effects not yet investigated. Also, pure MS must eventually fail at large enough polymer masses (and correspondingly higher z), at which neighboring peaks would no longer be discerned. In contrast, neither polydispersity nor loss of individual peak resolution presents a problem in our method. The various z-bands evidently become filled more homogeneously as the distributions become broader. However, Figure 2a shows that it is as easy to analyze narrow as wide mass distributions, since a greater loading of the z-bands does not broaden them. Loss of mass resolution arising at high m is not a problem either for the determination of mass distributions, as shown by our data going up to 140 kDa. The determination of mass distributions can be extended to higher m and z, as long as adjacent z-bands can be resolved, which is much easier than resolving individual ion peaks. Even when neighboring z-bands are no longer discernible, their position in the IMS-MS plane is theoretically known, so approximate mass distributions can still be obtained with no upper mass limit (beyond the usual MS limitations in spraying, transmitting, and detecting the ions). The mobility-mass relation for each band can be determined once for all and will be reproduced subsequently (except perhaps for the weak presence of extra conformers found in some samples). We have not studied mixtures of several homopolymers, but the bands associated with globules of different bulk densities would appear with different slopes in the IMS-MS spectra, and therefore they might be discriminated except at line crossings. If single peaks cannot be distinguished at these crossings, this would result in partial loss of information for some narrow m/z

Figure 3. Comparison between the minimal mass mmin(z) observed for samples of PEG 6k, PEG 12.6k, and PEG 14k as a function of the charge state z and the minimal mass m*(z) at which a globular structure would become unstable. 2714

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Table 1. Width of the pz(m) Distributions versus z, Defined in Terms of the Two Half-Height Masses, m+1/2 and m−1/2, at Which pz(m±1/2) = 1/2a

a

z

2

3

4

5

6

7

8

m+1/2/m−1/2

2.500

1.840

1.522

1.471

1.311

1.274

1.209

From the data of Figure 2d for PEG 14k.

more polar polymers. We have succeeded via negative ESI (NESI) in the case of PEG and anticipate comparable results for other polyethers. The general strategy to achieve globular ions relies on choosing the charging solution ion such as to avoid its strong binding to the polymer chains. Particularly interesting in the absence of specific ion binding sites is our quantitative confirmation that the charge retained by a polymer globule is determined by ion evaporation from its dry surface. This charge control mechanism had not been previously observed for polymer-ion combinations binding strongly. The ion evaporation mechanism yields an unusually simple and reproducible charging pattern, where each mass is present at most in two charge states. The resulting narrow mass range associated with a particular charge state offers some interesting advantages for mass analysis of polymers, particularly at high masses at which individual ion peaks are no longer recognizable in the mass spectrum.

ranges in the crossing regions (where only the sum of abundances of both polymers could be determined). However, some of the ions lost in these crossings will in general appear in a neighboring z-band which, given the reproducibility of the charge distributions pz(m), would allow recovery of the information lost. All of the advantages of our IMS-MS setup are presumably shared and may even be excelled by the more modern instrument of Bagal et al.,14 whose measurements extended only up to 6 kDa but who could perhaps have studied masses as large as those investigated here. The resolving power of our IMS is higher than theirs, and we enjoy the advantage of having a readily interpretable linear mobility measurement. However, we suffer from the complexity of some charge loss after the DMA measurement, apparently due to radiofrequency (RF) heating of the ions as they enter the MS. This inconvenience may perhaps be partly averted in the Synapt-based measurements of Bagal et al., since their mobility is determined in a relatively low-pressure region. However, because the whole ion path through the mobility cell in their instrument is subject to energizing RF fields (used for confinement), the real viability of this presumed advantage remains to be established. The method of charge reduction in the work of both Huang et al.15 and Bagal et al.14 is attributed to the addition of amine vapors. In reality, as shown by Larriba,12 this charge reduction requires both the presence of the amine as well as a considerable ion activation at the entry section of the MS. This activation provides added flexibility in the charge control process but leads to complications when applying the technique in other instruments or for other more labile chains. A substantial difference in charging patterns is clear, as our negatively charged ions appear at most in two charge states for a fixed mass, while the combination of amines and collisional activation leads to much broader charge distributions. Note in particular (Figure 2d) that the relative mass range spanned in negative ionization by a given z shrinks at increasing z. This point is quantified in Table 1. It is clear also from our raw IMSMS spectrum of Figure 2a, where the length of successive bands shrinks as one moves up in the IMS-MS plane. At large enough masses, the region of that plane occupied by a particular polymer will have shrunk to almost a line, greatly facilitating the process of reconstructing mass distributions with unresolved peaks, even for mixtures. This simplification is unlikely to arise in the amine-based charging process, where the final charge distribution is far broader and is not deterministically fixed by the charging mechanism, involving also the far less controllable process of collisional activation at the vacuum interface of the MS.

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ASSOCIATED CONTENT

* Supporting Information S

(1) Low z acquired by chains unable to bind ions, (2) charge state and shape assignment from mobility data, (3) analysis of narrow mass distribution PEG 12.6k, (4) multiple conformers, (5) charging probability at low z captured with PEG 6k, (6) more on the ionization mechanism, (7) measurements at reduced PEG concentration, and (8) study of a sample of PEG 2k. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): Following Yale rules, Juan Fernandez de la Mora declares that he has a personal interest in the company SEADM manufacturing the mobility analyzer used in this work.

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ACKNOWLEDGMENTS This work was partly supported by the U.S. AFOSR Contract FA9550-09-C-0178 to Alameda Applied Sciences, through a subcontract to Yale as part of a Phase II STTR program. We are grateful to Applied Biosystems/MDS Sciex and SEADM for the loan of the MS and DMA, respectively, and to the Keck Biotechnology Resource Laboratory at Yale for hosting the tandem instrument. We thank specially Dr. Carlos LarribaAndaluz for numerous fruitful comments on an earlier version of this manuscript and Dr. Bruce Thomson for his advice on MS matters.

6. CONCLUSIONS Our goal was to produce predominantly globular polymer ions of moderate charge states by ESI, in order to facilitate ion identification in IMS-MS measurements. Inspired by observations on the limited charge naturally acquired by polystyrene and other nonpolar polymer ions in ESI, we have sought to achieve similarly low charge states and globular geometries with

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REFERENCES

(1) Wong, S. F.; Meng, C. K.; Fenn, J. B. J. Phys. Chem. 1988, 92, 546−550. (2) Jackson, A. T.; Scrivens, J. H.; Williams, J. P.; Baker, E. S.; Gidden, J.; Bowers, M. T. Int. J. Mass Spectrom. 2004, 238, 287−297.

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(3) Becker, C.; Qian, K.; Russell, D. H. Anal. Chem. 2008, 80, 8592− 8597. (4) Robinson, E. W.; Garcia, D. E.; Leib, R. D.; Williams, E. R. Anal. Chem. 2006, 78, 2190−2198. (5) Trimpin, S.; Plasencia, M.; Isailovic, D.; Clemmer, D. E. Anal. Chem. 2007, 79, 7965−7974. (6) Song, J.; Grun, C. H.; Heeren, R. M. A.; Janssen, H. G.; van den Brink, O. F. Angew. Chem., Int. Ed. 2010, 49, 10168−10171. (7) Nasioudis, A.; Heeren, R. M. A.; van Doormalen, I.; de Wijs-Rot, N.; van den Brink, O. F. J. Am. Soc. Mass Spectrom. 2011, 22, 837−844. (8) Ude, S.; Fernandez de la Mora, J.; Thomson, B. A. J. Am. Chem. Soc. 2004, 126, 12184−12190. (9) Saucy, D.; Ude, S.; Lenggoro, W.; Fernandez de la Mora, J. Anal. Chem. 2004, 76, 1045−1053. (10) Ki, Ku, B.; Fernandez de la Mora, J.; Saucy, D. A.; Alexander, J. N., IV Anal. Chem. 2004, 76, 814−822. (11) Ude, S.; Fernandez de la Mora, J.; Alexander, J. N., IV; Saucy, D. A. J. Colloid Interface Sci. 2006, 293, 384−393. (12) Larriba, C. Production of ions and particles via simple and compound electrosprays in vacuum, gases or liquids (polar and non-polar). Ph.D. Dissertation, Yale University, New Haven, CT, 2010. (13) Larriba, C.; Fernandez de la Mora, J. J. Phys. Chem. B 2012, 116, 593−598. (14) Bagal, D.; Zhang, H.; Schnier, P. D. Anal. Chem. 2008, 80, 2408−2418. (15) Huang, L.; Gough, P. C.; DeFelippis, M. R. Anal. Chem. 2009, 81, 567−577. (16) Kaufman, S. L.; Skogen, J. W.; Dorman, F. D.; Zarrin, F.; Lewis, K. C . Anal. Chem. 1996, 68, 1895−1904. (17) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1998, 9, 957−965. (18) Pitteri, S. J.; McLuckey, S. A. Mass Spectrom. Rev. 2005, 24, 931−958. (19) De Winter, J.; Lemaur, V.; Ballivian, R.; Chirot, F.; Coulembier, O.; Antoine, R.; Lemoine, J.; Cornil, J.; Dubois, P.; Dugourd, P.; Gerbaux, P. Chem.Eur. J. 2011, 17 (35), 9738−9745. (20) Fenn, J. B.; Rosell, J.; Meng, C. K. J. Am. Soc. Mass Spectrom. 1997, 8, 1147−1157. (21) Chung, J. K.; Consta, S. J. Phys. Chem. B 2012, 116, 5777−5785. (22) Attoui, M.; Fernandez-García, J.; Cuevas, J.; Vidal, G.; Fernandez de la Mora, J. J. Aerosol Sci. 2013, 55, 149−156. (23) Rus, J.; Moro, D.; Sillero, J. A.; Royuela, J.; Casado, A.; Fernández de la Mora. Int. J. Mass Spectrom. 2010, 298, 30−40. (24) Hogan, C. J., Jr.; Fernández de la Mora. J. Phys. Chem. Chem. Phys. 2009, 11, 8079−8090. (25) Ude, S. Measurement and properties of nanometer particles in the gas phase. Ph.D. Dissertation, Yale University, New Haven, CT, 2004. (26) Larriba, C.; Hogan, C. J., Jr.; Attoui, M.; Borrajo, R.; Fernandez Garcia, J.; Fernandez de la Mora, J. Aerosol Sci. Technol. 2011, 45 (4), 453−467. (27) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60 (20), 2299− 301. (28) Karas, M.; Bahr, U. Trends Anal. Chem. 1990, 9 (10), 321−5. (29) Hall, Z.; Robinson, C. V. J. Am. Soc. Mass Spectrom. 2012, 23 (7), 1161−1168 ; see Figure 1c.

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dx.doi.org/10.1021/ac303054x | Anal. Chem. 2013, 85, 2710−2716