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

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  • Design of instrumentation for probing changes in electrospray droplets via theSternVolmer relationshipJason E. Ham, Bill Durham, and Jill R. Scott Citation: Review of Scientific Instruments 76, 014101 (2005); doi: 10.1063/1.1823191 View online: View Table of Contents: 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 SternVolmer 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 SternVolmer relationship. SternVolmer 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]


    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.1315However, 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),1822 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


    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 SternVolmerrelationship24 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,2529 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 SternVolmerrelationship, which shows promise for further exploration ofchanges in the chemical composition of electrosprayed drop-lets.


    A schematic diagram of the electrospray fluorescencesystem is provided in Fig. 1. This system was equipped witha Nd:yttriumaluminumgarnet(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),HeNe 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.3032

    F. Electrospray stability apparatus

    A HeNe 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 HeNe 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

    fRusbpyd3gCl26H2O (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).fRusbpyd3gCl26H2O 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 fRusbpyd3gCl26H2O. All samples wereinjected into the electrospray chamber through the 250mL...


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