Design of instrumentation for probing changes in electrospray droplets via theStern–Volmer relationshipJason E. Ham, Bill Durham, and Jill R. Scott Citation: Review of Scientific Instruments 76, 014101 (2005); doi: 10.1063/1.1823191 View online: http://dx.doi.org/10.1063/1.1823191 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/76/1?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Printing of organic and inorganic nanomaterials using electrospray ionization and Coulomb-force-directedassembly Appl. Phys. Lett. 87, 263119 (2005); 10.1063/1.2149985 A high-frequency electrospray driven by gas volume charges J. Appl. Phys. 97, 123309 (2005); 10.1063/1.1927279 Spraying modes in coaxial jet electrospray with outer driving liquid Phys. Fluids 17, 032101 (2005); 10.1063/1.1850691 Fine structure of the (S 1 ←S 0 ) band origins of phthalocyanine molecules in helium droplets J. Chem. Phys. 121, 9396 (2004); 10.1063/1.1804945 Comparison of size distribution of polystyrene spheres produced by pneumatic and electrospray nebulization AIP Conf. Proc. 550, 322 (2001); 10.1063/1.1354419

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Design of instrumentation for probing changes in electrospray dropletsvia the Stern–Volmer relationship

Jason E. Ham and Bill DurhamDepartment of Chemistry and Biochemistry, University of Arkansas, 10l Chemistry,Fayetteville, Arkansas 72701

Jill R. Scotta)

Idaho National Engineering and Environmental Laboratory (INEEL), 2525 North Fremont Avenue,Idaho Falls, Idaho 83415

(Received 28 April 2004; accepted 26 September 2004; published online 17 December 2004)

Electrospray ionization(ESI) is a proven method for introducing large intact molecules into the gasphase. However, the processes that occur within this ion source are poorly understood. We havedeveloped instrumentation and methodology to probe the evolution of droplets within theelectrospray plume. Using emission lifetime spectroscopy, excited-state lifetimes offRusbpyd3g2+

with and without a known quencher, 2,3,5,6-tetramethyl-p-phenlyenediamine, present wereobserved. Lifetimes were shown to decrease as quencher concentration increased, as expected. Rateconstants(with and without quencher present) were determined and correlated with quencherconcentration using the Stern–Volmer relationship. Stern–Volmer plots reveal the linearity of thequenching reaction and can be used to determine the concentrations of species within theelectrosprayed droplets. The evolution of the ESI droplets can be probed by comparing theconcentration of a species at different locations within the plume. ©2005 American Institute ofPhysics.[DOI: 10.1063/1.1823191]

I. INTRODUCTION

Electrospray ionization(ESI) has become a well-integrated tool for mass spectrometry because of its applica-bility to many different analyte types. It has been used suc-cessfully in producing gas-phase ions of peptides,1,2

proteins,3,4 polymers,5 organometallics,6,7 and inorganics.8,9

However, the processes and mechanisms that must occur forproduction of these gas-phase stable ions from bulk solutionare inadequately understood.10 The problem stems from lim-ited technology and/or methodologies available to observethe formation of submicron droplets from solution, and moreimportantly, how the relevant concentrations of analyte inthese droplets change during evolution of the electrosprayplume.

ESI is different from other ionization schemes in that thecharge on the analyte is either already present, such as forinorganic salts, or induced by evaporation of solvent anddeposition of charge as seen with neutral organic com-pounds. Therefore, it is not surprising that early investiga-tions of the electrospray process have shown that the massspectra from ESI and its ionization efficiency are stronglyinfluenced by the solution content.11,12 This dependency isderived from thepH, buffer, and compositional changes ofmixed solvents that occur within the droplets as theyevolve.13–15However, the extent of these effects is difficult toquantify due to the complex dynamics of the spray process.At early stages of the ESI process, electrochemical reactions

within the emitting capillary can change the solution compo-sition. Bladeset al.16 have shown that Zn+2 and Fe+2 can bereleased into the solution by the oxidation of the electrosprayneedle. Additionally, electrophoretic charge separation ofspecies in the needle can also alter the solution content. Zhouand Cook have shown that this electrophoretic mechanismmay be responsible for anomalous sensitivity variations ob-served in electrospray mass spectra.17 Other attributes of theelectrospray process that contribute to the solution dynamicsare uneven droplet fission and solvent evaporation. All ofthese factors make describing a detailed mechanism quitechallenging.

Most optical studies designed to probe the ESI plumehave employed laser-induced fluorescence spectroscopy(LIFS),18–22 although absorption spectroscopy has also beenused.23 Due to its high sensitivity, LIFS has been shown to bea good approach for determining solute and solvent changesin the submicron size droplets. Zhouet al.18 used this tech-nique to probe the solvent fractionation in electrospray drop-lets using the solvatochromic dye, Nile Red. These studiesshowed that the polarity of the droplet increases as the drop-lets move away from the electrospray needle. In a similarstudy, Zhouet al.19 also used this technique to monitorpHsensitive dyes in ESI droplets. These studies showed how thepH decreases as a function of distance from the electrosprayneedle to an extent greater than expected from solventevaporation alone. All of these studies have relied on theinteraction of the fluorophore with the solvent to explore theESI process.

An alternative method for probing the composition ofelectrosprayed droplets using LIFS is to add quenchers to the

a)Author to whom correspondence should be addressed; electronic mail:scotjr@inel.gov

REVIEW OF SCIENTIFIC INSTRUMENTS76, 014101(2005)

0034-6748/2005/76(1)/014101/6/$22.50 © 2005 American Institute of Physics76, 014101-1

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solution. Addition of quenchers that deactivate the emissionof the analyte provides a way to survey the concentrations ofspecies in the electrospray plume. By monitoring the emis-sion decay signals, the rates of the bimolecular interaction ofa fluorophore and quencher can be ascertained. This colli-sional quenching can be described by the Stern–Volmerrelationship24 and used to determine the concentrations of thequencher as the droplets evolve. This method is dissimilar tothe previous studies, which relied on the dynamics of thesolvent.

In this article, we describe an instrument and method toprobe changes in the chemical components of electrospraydroplets. Using methodology developed from our experiencein ruthenium chemistry,25–29 we have developed a way tomonitor the quenching reactions offRusbpyd3g2+ with2,3,5,6-tetramethyl-p-phenylenediamine(TMPD) duringdroplet evolution. The observed quenching reactions in theESI plume are shown to correlate with the Stern–Volmerrelationship, which shows promise for further exploration ofchanges in the chemical composition of electrosprayed drop-lets.

II. INSTRUMENT DESIGN

A schematic diagram of the electrospray fluorescencesystem is provided in Fig. 1. This system was equipped witha Nd:yttrium–aluminum–garnet(Nd:YAG) (third harmonic,355 nm) laser for excitation of droplets formed in the elec-trospray chamber. A glass slide placed in front of the laseracted as a beam splitter to reflect a small portion of light to adiode trigger, which triggered the oscilloscope for data col-lection. As the light entered the chamber, it irradiated drop-lets formed by the electrospray process that containedfRusbpyd3g2+. The relatively long-livedfRusbpyd3g2+ excitedstates emit light atl.550 nm. A photomultiplier collectedthe emitted light and the resulting signal was amplified andsent to the input of an oscilloscope. All data from the oscil-loscope were then transferred to a computer for analysis.

A. Electrospray chamber

The electrospray chamber(shown in Fig. 2) was con-structed of an aluminum boxs5.5 in.35.5 in.310.25 in.dwith an aluminum bases7 in.35.5 in.d. Each of these sides

is 0.25 in. thick and painted internally flat black to reducereflections. The top enclosure is equipped with a micrometerdriven horizontal translator[Fig. 2(d)] and photomultipliertube [Fig. 2(e)]. The translator allows the photomultipliertube to be moved with the excitation laser beam along thexaxis to improve optical detection.

B. Syringe pump and sample delivery

Samples were delivered to the electrospray chamber by aBaby Bee syringe pump(Bioanalytical Sciences Inc., WestLafayette, IN). The pump[Fig. 2(a)] is used to push solutionfrom a 250mL Hamilton Syringe(Gastight No. 1725, Reno,NV) into the electrospray needle. The end of the Hamiltonsyringe was inserted into an injection fill port(Valco Instru-ments Inc., Houston, TX) which is connected to a 7 in. pieceof PEEK tubing[inner diameter(i.d.) =0.508 mm, outer di-ameter(o.d.) =1.587 mm] (Upchurch Scientific, Oak Harbor,WA) for an overall dead volume of approximately 36mL.

C. The needle and counter-electrode

The electrospray needle[Fig. 2(g)] was constructed oftwo stainless steel syringe needles. The inner needle(30gauge, i.d. =152mm, o.d. =309mm] (Small Parts Inc., Mi-ami Lakes, FL) was inserted into a 23 gauge needle(i.d.=330mm, o.d. =635mm) (Becton Dickson & Co., FranklinLakes, NJ) and then soldered using 60/40 tin/lead rosin-coresolder. The needle was electrically connected to earth groundthrough the electrode chamber. The counter-electrode[Fig.2(h)] was machined from solid brass to a diameter of 1 in.and center-drilled for a 0.25 in. diameter hole to hold a glasscapillary for support. During all experiments, +5.5 kV wassupplied to the counter-electrode via a high voltage directcurrent power supply(EH Series, Glassman High VoltageInc., Whitehouse Station, NJ).

D. Excitation source

Aerosol droplets formed by the electrospray source wereprobed using the third harmonic of a Nd:YAG DCR-1 laser(Quanta Ray, Mountain View, CA) with a pulse width at halfheight of 10 ns at 355 nm. The output power of the laser wasmaintained at the minimum power necessary to obtain a rea-sonable signal. The excitation beam was focused using a

FIG. 1. Schematic of the electrospray fluorescence system showing relativelayout and connectivity of hardware: A Nd:YAG laser beam(a) passesthrough a beam splitter glass slide(b) which directs light to a diode trigger(c). Aerosol droplets are formed in the ESI chamber(d). Fluorophores in thedroplets are then excited and detected by a photomultiplier tube(e) whoseoutput is driven by an amplifier(f) into an oscilloscope(g). Data from theoscilloscope are transferred to a computer(h) for analysis.

FIG. 2. Schematic of the electrospray chamber showing syringe pump(a),He–Ne laser(b), photomultiplier tube(c), translational stage(d), focus lens(e), and iris (f). Top inset schematic shows magnified view of the electro-spray needle(g) and counter-electrode(h).

014101-2 Ham, Durham, and Scott Rev. Sci. Instrum. 76, 014101 (2005)

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2 in. lens sF.L. =1500 mmd through a 2 in. iris opened to5 mm. This produced a 2 mm diameter spot size at a dis-tance of 10 mm from the end of the electrospray needle.

E. Optical detection and analysis

Emission from the electrosprayed droplets was collectedwith a 1 in. diameter focusing lens with a focal length of5 cm and passed through a 550 nm short wavelength cutofffilter (Esco Products Inc., Oak Ridge, NJ) and finally de-tected with a R928 photomultiplier tube(Hamamatsu Photo-nics K.K., Toyooka Village, Japan). Signal was then fed intoa DC-300 MHZ amplifier (Stanford Research Systems,Sunnyvale, CA) set at 50V to drive the input for a Lecroy7200 oscilloscope(Lecroy Corp., Chestnut Ridge, NY). Alldata collected on the oscilloscope were transferred to a com-puter and analyzed. The interface and data analysis softwarehas been previously used for solution laser flash photolysisexperiments.30–32

F. Electrospray stability apparatus

A He–Ne laser(model 1101, Uniphase, Sunnyvale, CA)was also added to the system[Fig. 2(b)] to monitor the sta-bility of the electrospray source. By moving the He–Ne laseralong thex axis, away from the needle, the evolution ofdroplets into the gas phase could be observed as seen inpictures taken with a 403 charge coupled device(CCD)camera (Edmund Optics Inc., Barrington, NJ) in Figs.3(a)–3(e).

G. Chemicals and sample preparation

fRusbpyd3gCl2·6H2O (where bpy 5 bipyridine) waspurchased from Sigma-Aldrich(St. Louis, MO). Acetonitrile(HPLC grade) was purchased from EMD Chemicals(Gibbs-town, NJ). Milli-Q water s18 MVd was provided by a Milli-pore purification system(Billerica, MA). TMPD was pur-chased from Aldrich Chemical Co.(Milwaukee, WI).fRusbpyd3gCl2·6H2O was dissolved in a 95%acetonitrile/5% Milli-Q water solution to a final concentra-tion of 25 mm. For quencher experiments, TMPD was addedto produce final concentrations of 1, 2, 4, 5, and 6 mMTMPD with 26 mM fRusbpyd3gCl2·6H2O. All samples wereinjected into the electrospray chamber through the 250mLsyringe at a flow rate of 5mL/min.

III. RESULTS AND DISCUSSION

A detailed drawing of the hardware and connectivity ofthe overall electrospray apparatus is shown in Fig. 1, anddetails of the electrospray chamber, where optical detectionis observed, are given in Fig. 2. To obtain the lifetime data, aphotomultiplier tube(PMT) was chosen as the optical detec-tor. This is similar to instrumentation employed by Ideue22

and Chillier,23 instead of a Dilor XY Raman spectrometerused by Zhouet al.18–20

Before performing the LIFS experiments, the uniformityof the electrospray was ascertained by visually inspectingstop-action photographs as shown in Fig. 3. The photographswere obtained by placing a 30 mW He–Ne laser perpendicu-lar to the electrospray needle and a CCD camera inserted at a

90° angle above the electrospray needle. Photographs oflight scattering from the electrosprayed droplets were thenobtained at increasing distances between the tip of the needleand the laser beam. The light scattering intensity is a factorof the expansion of the plume and the size of the droplets. InFig. 3(a), the formation of the Taylor cone33 at the end of theelectrospray needle is observed along with an immediate ex-pansion of the ESI plume. A subsequent decrease in the in-tensity is then seen as droplets in the plume decrease in sizewith increasing distances from the needle as illustrated inFigs. 3(b)–3(e). Comparison of the photographs in Fig. 3with photos published in the literature,34,35 confirm that thespray is uniform. The data taken below were obtained at adistance of 10 mm from the spray, which corresponds to Fig.3(e).

Olumeeet al.36 have employed phase Doppler anemom-

FIG. 3. Stopped-action photographs of aerosol droplets formed in the elec-trospray chamber. Pictures show the evolution of electrosprayed droplets asa function of increasing distances of 2, 4, 6, 8, and 10 mm for(a)–(e),respectively.

014101-3 Probing ESI Droplets via Stern–Volmer Rev. Sci. Instrum. 76, 014101 (2005)

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etry to determine the axial velocities of droplets in an elec-trospray plume. They found that at approximately 10 mmfrom the needle, using a 90% methanol/10% water mixtureand a 4 kV onset voltage, the average velocity is,10 m/s.Since fRusbpyd3g+2 has an emission lifetime of approxi-mately 400 ns, this would indicate that a droplet would moveabout 4mm during a lifetime experiment. Because the exci-tation laser source has a beam diameter of 2 mm, the dropletwould seem to be motionless within the time limits of theexperiment. Additionally, the miniscule motion of the drop-lets also ensures that they remain within the relatively largelight collecting area of the PMT.

Emission lifetime spectroscopy is a useful tool for deter-mining rates of electron transfer with a variety of chemicalspecies. The scheme in Fig. 4 illustrates the general series ofreactions thatfRusbpyd3g2+ undergoes with a reductivequencher,Q. The excited statesRuII* d normally decaysthrough a number of deactivation pathways back to theground state governed by the overall rate constantskdd. Thisrate constant is easily measured using emission lifetimespectroscopy in the absence ofQ. When a quencher such asTMPD is added, RuII* can accept an electron to form RuI

leaving the quencher with a +1 charge. This overall processdecreases the emission lifetime of the excited statesRuII* d.Figure 5 shows the emission lifetimes offRusbpyd3g2+ withincreasing concentrations of 2,3,5,6-tetramethyl-p-phenylenediamine, a known quencher of the ruthenium ex-cited state. All lifetimes with the excitation beam were di-rected at a point 10 mm from the electrospray needle. Asseen in the graph, the emission lifetimes were observed todecrease with increasing concentrations of the quencher. At0 mM TMPD, the observed rate constant for decay of theemission offRusbpyd3g2+ was 4.803106 s−1 and increased to9.693106 s−1 with 6 mM TMPD.

From these quencher experiments, plots of thekobs forthe emission decay versus the quencher concentration weremade. These were fitted to the Stern–Volmer equation[Eq.(1)], wherekobs is the observed rate constant,kR is the rate

constant for radiative decay,kNR is the inclusive rate constantfor all nonradiative processes,kq is the bimolecular quench-ing constant, andQ is the quencher concentration. The formof this equation was chosen to avoid unreliable absorbancecorrections due to added quencher

kobs= kR + kNR + kqfQg. s1d

In this equation, the sumkR+kNR is equivalent tokd inthe scheme in Fig. 4, which is determined in experimentswith no quencher present.

Because TMPD has a weak emissionst1/2,50 nsd whenexcited at 355 nm that coincides with the emission offRusbpyd3g2+, its contribution to the overall fluorescence sig-nal must be considered before accurately fitting to the Stern–Volmer relationship. Solutions containing 1–6 mM TMPDonly were made and sprayed using the same conditions assamples that also containedfRusbpyd3g2+. The fluorescentsignals from the TMPD-only solutions were then subtractedfrom those signals obtained withfRusbpyd3g2+ present. Thiseliminated the emission contribution of the TMPD to theemission lifetime.

The observed rate constantsskobsd for the emission life-times are listed in Table I and plotted in Fig. 6 for data fromdistances of 6 and 10 mm. The Stern–Volmer plot createdfrom the rate constants obtained versus the concentration ofTMPD show that they are, indeed, directly proportional asseen by the good linear fit and correlation factors of 0.942and 0.945 for 6 and 10 mm, respectively. The standard de-viations of the points in this graph were less than 5%. Theslope of a line gives the apparent quenching constantskqd.The quenching constants are 2.83105 and 7.13105 mM−1

obtained at 6 and 10 mm, respectively. These values are con-

FIG. 4. Schematic illustrating formation and reaction of excited state withquencher.

FIG. 5. Graph of the emission lifetimes offRusbpyd3g2+ with increasingconcentrations of TMPD.

TABLE I. ObservedfRusbpyd3g2+ emission rate constantsa and calculatedconcentrationsb of TMPD for droplets at 6 and 10 mm are listed accordingto the initial bulk concentration of TMPD.

6 mm 10 mm

Bulk[TMPD] kobs [TMPD] kobs [TMPD]

0 4.803106 4.803106

1 5.293106 1.77 6.533106 2.432 5.593106 2.86 7.283106 3.494 5.963106 4.17 8.053106 4.585 8.753106 5.566 9.693106 6.88

aRate constants given in units of s−1.bConcentration given in units of mole/liter.

FIG. 6. Stern–Volmer plot offRusbpyd3g2+ with TMPD.

014101-4 Ham, Durham, and Scott Rev. Sci. Instrum. 76, 014101 (2005)

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siderably smaller than the value of the quenching constantreported in the literature,37 kq=1.23107 mM−1 obtainedfrom bulk solution data. The discrepancy between the bulkand droplet quenching constants is not thoroughly under-stood. One of the factors that may contribute to this differ-ence is the electrophoretic migration of species within theESI needle, which is known to alter the solution content.33

While this migration is not expected to affect the neutralTMPD, it may impact the actual concentration offRusbpyd3g2+ in the droplets. A thorough understanding ofthe phenomena giving rise to the discrepancy requires moreextensive studies that are beyond the scope of this article.However, phenomena that occur after the droplets have beenformed can be addressed.

By rearranging Eq.(1) and including the relationshipthat kd=kR+kNR, the concentration of the quencher in thedroplets can be calculated according to Eq.(2):

fQg =skobs− kdd

kq. s2d

The concentrations of TMPD calculated using Eq.(2) aregiven in Table I. Although the changes in concentrations be-tween the bulk solution and formation of the droplets is stillnot thoroughly understood, the change in concentration ofthe quencher that occurs after the droplets are formed doesprovide insight into the evaporation process of ESI.

The increase in concentration of the quencher from the6 mm distance to the 10 mm location is primarily due toevaporation of the volatile solvent acetonitrile. The evapora-tion of acetonitrile by itself does not significantly change therate constants for the emission decay offRusbpyd3g2+ be-tween 6 and 10 mm(Table I) when no quencher is present.Because the evaporation process is accompanied by coolingand fRusbpyd3g2+ emission is known to vary withtemperature,38 the temperature change in the droplets be-tween 6 and 10 mm is also negligible or counteracted bysome other factor(s). The most significant factor appears tobe the change in quencher concentration. The evaporation ofacetonitrile causes an average concentration change of0.57 mM of TMPD between droplets at 6 and 10 mm.

If it is assumed that the average number of moles ofTMPD does not change as the droplets progress from 6 to10 mm, which is reasonable given that TMPD is a solid atroom temperature, then the ratio of the concentration changeis indicative of the percent change in volume of the droplets.For example, for solutions sprayed from a bulk concentrationof 4 mM, the ratio of[TMPD] at 6 mm divided by that at10 mm is 0.91. Therefore, the average volume of the dropletsat 10 mm is 91% less than that at 6 mm for droplets initiallyformed from 4 mM bulk solution. Interestingly, the percentvolume change appears to depend on the initial bulk concen-tration as the volume at 10 mm is 73%, 82%, or 91% that ofthe volume at 6 mm for initial bulk concentrations of 1, 2,and 4 mM, respectively. This suggests that the initial concen-tration of TMPD may affect the rate of acetonitrile evapora-tion. Perhaps TMPD increases hydrogen bonding or van derWaals forces that retard the evaporation of acetonitrile; sothat the larger the concentration of TMPD, the slower therate of acetonitrile evaporation. It is not surprising that addi-

tional concentration of a solution component could changethe rate of evaporation because its presence would affect thesurface tension of the droplets. The rate of evaporation canbe estimated by assuming that the average velocity of thedroplets is ,10 m/s as determined by Olumee andco-workers.36 Therefore, it takes the droplet approximately0.0004 s to cross the 4 mm distance between the 6 and10 mm observation points. Therefore, the evaporation ratesin terms of percent change per second are 6.753104, 4.503104, and 2.253104%s−1 for initial bulk TMPD concentra-tions of 1, 2, and 4 mM, respectively. From these percentchange rates, it can be estimated that 100% of the acetonitrilesolvent should evaporate in 1.5310−3, 2.2310−3, and 4.4310−3 s for bulk [TMPD] of 1, 2, and 4 mM, respectively.Therefore, under the conditions of these experiments, fullydesolvated ions should be formed after,5310−3 s. Theseexperiments and calculations give a glimpse into the changein droplet volumes and solvent evaporation mechanism in-volved in ESI.

By using the instrumentation along with the methodol-ogy of ruthenium-quencher chemistry, the evolution of elec-trosprayed droplets can be explored using the Stern–Volmerrelationships at varying distances from the needle. This willallow the processes and mechanisms for the formation offully desolvated ions from solution droplets to be investi-gated, which will then provide a better understanding of theunderlying principles of electrospray ionization. Such futureexperiments may help to define the extent to which relativeconcentrations of species in electrosprayed droplets affectthe mass spectra and ionization efficiency.

ACKNOWLEDGMENTS

J.E.H. would like to thank the INEEL ACE Fellowshipprogram for funding. The authors also gratefully acknowl-edge support from the United States Department of Energy,under Contract No. DE-AC07-99ID13727 BBWI.

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014101-6 Ham, Durham, and Scott Rev. Sci. Instrum. 76, 014101 (2005)

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