Fluorescence and Electronic Action Spectroscopy of Mass-Selected

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Fluorescence and Electronic Action Spectroscopy of Mass-Selected Gas-Phase Fluorescein, 2′,7′-Dichlorofluorescein, and 2′,7′Difluorofluorescein Ions Huihui Yao and Rebecca A. Jockusch* Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada S Supporting Information *

ABSTRACT: 2′,7′-Dichloro- and 2′,7′-difluorofluoresceins are superior alternatives to underivatized fluorescein. Although several studies characterizing their condensed-phase photophysical properties have been reported, little is known about their intrinsic characteristics. Here, the gas-phase properties of three charge states of each fluorescein are characterized using a quadrupole ion trap mass spectrometer which has been modified for spectroscopy. Electronic action spectra, constructed by monitoring the extent of photodissociation as a function of excitation wavelength, indicate that the gaseous dianions and cations resemble their solution-phase counterparts. In contrast, a large shift in the electronic action spectra of the monoanions indicates the presence of a different tautomer in the gas phase than that present in solution. The gaseous monoanion is deprotonated on the xanthene ring, rather than being deprotonated on the pendant group as found in soluion. The dianions and cations do not emit detectable fluorescence in the gas phase. In contrast, the monoanions do fluoresce, but the emission intensity is low and the spectra are broad. This work illustrates the effect of halogenation on the intrinsic properties of the dyes and provides useful fundamental understanding that promises to aid the development more robust fluorescent dyes.

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owing to its bright fluorescence and ease of conjugation to biomolecules. Despite its popularity, FL undergoes photodegradation and its fluorescence and absorption characteristics have a strong dependence on solvent7 and pH.8 To reduce those drawbacks, FL derivatives, 2′,7′-difluorofluorescein (DFF, also known as Oregon Green 488)9 and 2′,7′-dichlorofluorescein (DCF)10 were developed (Scheme 1). DFF and DCF are both significantly more photostable than underivatized fluorescein.9,11−13 Another advantage of these halogenated derivatives is that the pKa of DFF and DCF are 4.95 and 4.69 in aqueous solution, values which are much lower than that of FL (6.44).14 Hence, the charge state and thus the fluorescence and absorption properties of DFF and DCF are less sensitive to the solvent media in the physiological pH range.11,12 DFF and DCF constructs have been employed in a wide variety of applications, including fluorescent protein labeling and imaging11,15,16 as acid−base indicators,17 fluorescent Ca2+ indicators,11,17,18 a Zn2+ indicator,12 as well as in measuring oxidative stress.19,20 Owing to the widespread usage of FL and its derivatives, extensive studies have been carried out to investigate their important chemical properties such as

INTRODUCTION Fluorescence spectroscopy is one of the most powerful analytical techniques owing to its superior detection limit, which can be as low as a single molecule and, for many fluorophores, the high sensitivity to the environment in which the analyte is embedded. Several different measurables provide information in fluorescence experiments, including emission intensity, spectral features, and fluorescence lifetime. Fluorescence is widely used in highly diverse scientific fields, including physics, chemistry, life sciences, and mineralogy.1 In the last few decades, much effort has been put into the development of fluorescence-based techniques for the study of the conformation and dynamics for large biomolecules. These techniques include Fö r ster resonance energy transfer (FRET), 2,3 fluorescence lifetime imaging microscopy (FLIM),4,5 and so on. In our laboratory, fluorescence spectroscopy is combined with another powerful technique, mass spectrometry.6 When used in combination with a mass spectrometer, fluorescence spectroscopy can provide quantitative measurements on well-defined species of a single m/z value that is mass-selected in the mass spectrometer. Thus, any ambiguity is eliminated as to which species in a mixture is contributing to the fluorescence signal, which can be an issue in solution. Moreover, intrinsic properties of the analyte of interest can be investigated in the absence of solvent molecules. Fluorescein (FL) is a well-known fluorescent probe that has been used widely in biological and biochemical applications © 2013 American Chemical Society

Special Issue: Peter B. Armentrout Festschrift Received: October 2, 2012 Revised: January 14, 2013 Published: January 16, 2013 1351

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Scheme 1. Molecular Structure of Fluorescein-Based Dye Cation, Monoanions, and Dianion: R = H, M = FL; R = F, M = DFF; R = Cl, M = DCF

to those of FL. In addition, fluorescence spectra of massselected isolated FL, DFF and DCF monoanions are presented for the first time.

ionization constants,21−23 excitation and emission properties in solution24 and excited-state proton transfer mechanism24−27 and so forth. FL, DFF, and DCF in aqueous solution can exist in four prototropic forms and up to seven tautometric forms.8 The charged forms are shown in Scheme 1. Absorption of aqueous DCF and DFF varies significantly in different protonation (charge) states and these charge states show similar spectroscopic properties to those of FL. The dianions are the most fluorescent, with reported quantum yield of 0.90,7 0.97,28 and 0.9429 for FL, DFF, and DCF, respectively. The dianions each exhibit a single absorption maximum in the visible, which lies at 490 nm for FL7,30,31 and DFF24 and is red-shifted to 502 nm for DCF32 in aqueous solution. The monoanionic species have two absorption maxima in the visible, each with similar absorptivities. For FL,7,30,31 and DFF,24 these lie at 450 and 470 nm, whereas for DCF32 they are red-shifted to 465 and 490 nm. Despite the large amount of data available for FL, DFF, and DCF in solution, fluorescence of these dyes isolated in the gas phase, where microenvironmental effects presented by solvent molecules are absent, has not been extensively reported. Our group has previously shown that the dianion, which has a quantum yield close to unity in solution, is not fluorescent in vacuo.33 The dianion instead detaches an electron upon electronic excitation. The electronic action spectra of gaseous FL, constructed by monitoring photodissociation yield as a function of excitation wavelength, showed that the dianion and the cation have spectra similar to those of FL in solution; however, the monoanion exhibits a large red shift in its absorption maximum compared to that in solution.33 We suggested that the reason for this large shift is that a different tautomer of the monoanion (Mono_X, Scheme 1, which is deprontonated on the xanthene ring system) is preferred in the gas phase than that present in solution (Mono_XH, Scheme 1, deprotonated on the pendant carboxy phenyl ring).33 This suggestion is supported by results from infrared multiple photon dissociation (IRMPD) experiments34 and electronic structure calculations.34,35 Similar conclusions were also made from IRMPD experiments and computation on DCF.34 Very recent work measuring photodissociation spectra of FL monoanions in an electrostatic storage ring reports a large difference in electronic spectra measured at short times and with a 2 s delay added prior to laser irradiaton.36 This change was interpreted as conversion from the solution-type Mono_XH tautomer to the Mono_X tautomer preferred in the gas phase. Here, a quadrupole ion trap mass spectrometer is used to isolate individual charge states of halogenated fluorescein derivatives and separately probe their gas-phase spectroscopic properties. Electronic action spectra of the three charge states of DFF and DCF in the gas phase are presented and compared

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EXPERIMENTAL METHODS FL and DCF were purchased from Sigma-Aldrich (Oakville, Ontario) and DFF was purchased from Invitrogen (Burlington, Ontario). They were used without further purification. Two micromolar solutions for electrospray ionization (ESI) were prepared by dissolving FL, DFF and DCF in a 30:70 methanol−water mixture. In the studies of the dianions, a few drops of ammonium hydroxide were added to the solutions to increase the basicity of the electrospray solution. The solutions were electrospray ionized into a commercial quadrupole ion trap (QIT) mass spectrometer (Bruker Esquire 3000+, Bruker Daltonics Gmbh, Bremen, Germany), which has been modified for spectroscopy.6 In the QIT, protonated or deprotonated molecular ions of m/z ratios corresponding to the cationic, monoanionic, or dianionic forms were isolated, stored, and irradiated with frequency-doubled light from a pulsed tunable titanium:sapphire laser (Nd:YVO4 pumped Tsunami, Spectra-Physics Division, Newport Corp., Mountain View, CA. 80 MHz repetition rate, ∼130 fs pulse duration). The 1/e2 diameter of the laser beam is ∼700 μm at the center of the ion trap. The spatial extent of the cloud of trapped ions can be adjusted using the qz trapping parameter and is normally somewhat larger than the beam. However, with this arrangement ion populations can be completely dissociated because the trapped ions cycle at radio frequencies, passing in and out of the laser beam over the course of the experiment.6 The laser irradiation power is controlled by attenuation with a variable neutral density filter. Photodissociation of selected fragment ions was investigated to gain additional insight into the fragmentation pathways of the precursor ions. For these studies, fragments of the precursor ions were formed using collision-induced dissociation (CID). Fragments were subsequently isolated and irradiated with the same excitation wavelength and power used in photodissociation studies of their precursor ions, detailed below. Gas-phase Electronic Action Spectroscopy. For each photodissociation experiment, the ion accumulation time was adjusted to maintain an ion charge control (ICC) value for each charge state to be 35 000 ± 5000. This ensures that a similar number of ions are trapped and irradiated in each experiment, as the ICC value reflects the total ion current measured by the mass spectrometer. Helium bath gas pressure in the trap was maintained at the recommended value for the Bruker Esquire, which we estimate to be ∼1.3 × 10−6 bar.37 The photodissociation yield was measured as a function of excitation wavelength in the range of 410 to 540 nm to produce the action spectra. The photodissociation yield was taken as the 1352

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Figure 1. Photodissociation mass spectra of monoisotopically isolated DFF (a, b, c) and DCF (d, e, f) cations in blue (top, 420 nm excitation), monoanions in green (middle, 520 nm excitation), and dianions in red (bottom, 505 nm excitation). The qz value of the precursor ion is 0.27. Irradiation power and fragmentation time are chosen to show approximately 40−60% dissociation of precursor ions. Stars mark the precursor ion and arrows indicate dissociation pathways.

time of laser irradiation, respectively. In the power dependence studies, the laser irradiation time and qz were 50 ms and 0.27, respectively, for all charge states of FL, DFF, and DCF. In the kinetics studies of the monoanions, the power used was 2.5 mW. For both kinetics and power dependence experiments, the excitation wavelengths used were 500, 525, and 430 nm for the dianions, monoanions, and cations, respectively. Analyses of these data were aided by plotting the natural logarithm of the fraction of precursor ion remaining as a function of the irradiating power or time. Fluorescence Spectroscopy. Experimental conditions for the emission studies were selected to maximize the density of the trapped ions. ICC of the monoanionic forms of FL, DFF and DCF was ∼400 000. Ion qz value was set at 0.59 to increase the overlap between the ion cloud and the laser beam. The background helium pressure was increased to ∼1.9 × 10−6 bar to suppress photodissociation. Monoanions of FL, DFF, or DCF were stored in the QIT and exposed to 510 nm laser irradiation for 800 ms. The power of the laser beam was 1.0 mW going into the QIT. A 515 nm long pass filter was used to eliminate scatter from the excitation beam. A small portion of the emitted fluorescence was collected orthogonally to the path of the excitation beam, through a 2 mm diameter hole on the ring electrode. The detector used for fluorescence measurements is a spectrograph coupled to an electron-multiplying charge-coupled device (EM-CCD) camera (Shamrock 303i/ Newton, Andor Technology PLC, Belfast, Northern Ireland). One acquisition consisted of 500 scans of 80 ms each. The ICC normalized fluorescence spectra of FL, DFF, and DCF were compared with that of rhodamine 640 precursor ions (m/z 491.7), excited at the same wavelength, under the same experimental conditions. Reference 11 explains the experimental setup used for steady-state fluorescence measurements in greater details. Fluorescence lifetimes of the monoanions were measured by time-correlated single photon counting using a single photon

ratio of the decrease in precursor ion intensity upon laser irradiation normalized to the precursor ion intensity without laser irradiation: (Iprecursor,laser off − Iprecursor,laser on)/Iprecursor,laser off. Error bars correspond to the standard deviation of three sets of measurements taken on three different days. Parameters used for photodissociation were adjusted according to the stability of the precursor ion; dianions required the least energy to photodissociate whereas the cations required the most. The QIT trapping parameter qz was set to the standard value of 0.27 for the dianions and the monoanions, whereas it was increased to 0.59 for the cations to increase the irradiation efficiency of the trapped cations. The laser irradiation times used were 40, 100, and 100 ms for the dianions, monoanions, and cations, respectively. The laser power used for photodissociation was the lowest for the dianions (0.5, 1.2, and 0.8 mW for FL, DFF, and DCF, respectively) and highest for the cations (20 mW for FL, DFF, and DCF). For all three monoanions, a laser power of 5 mW was used. Due to the limitation of the power output from the frequency-doubled laser above 520 nm, a somewhat reduced laser irradiation power (2.4 mW) was used to repeat the experiment in the wavelength range of 500 to 540 nm for DFF and DCF monoanions. Solution-Phase UV/Visible Absorption Spectroscopy. Solution-phase UV/visible absorption spectra were measured on a Perkin-Elmer Lambda 12 UV/visible spectrometer. DFF and DCF powders were dissolved in water and the pH values of the aqueous solutions were adjusted with hydrochloric acid or ammonium hydroxide. The absorption spectra of 100 μM solutions of DFF and DCF ions were measured at pH 0.8 and 0.5 for the cations, at pH 3.9 and 3.8 for the monoanions (a pH at which neutrals are also present) and at pH 11.7 and 10.6 for of the dianions. Photodissociation Power Dependence and Kinetics. In the power dependence and kinetics studies, the fraction of precursor ion remaining after laser irradiation, calculated from Iprecursor,laser on/Iprecursor,laser off, was plotted against the power and 1353

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Figure 2. Electronic action spectra of gaseous FL (top), DFF (middle), and DCF (bottom) for the mass-selected cation (left column, blue squares), monoanion (middle column, green diamonds), and dianion (right column, red circles). The data points are connected by smooth dashed curves as a guide to the eye. UV/visible absorption spectra of FL, DFF, and DCF in aqueous solution of low (left), intermediate (middle), and high (right) pH are shown in solid lines normalized with the highest absorbance scaled to maximum photodissociation yield in the electronic action spectrum. Insets show electronic action spectra of the monoanions in the range 520−540 nm using approximately half of the excitation power used in the main monoanion spectra.

numerous other photodissociation fragments of the cations are observed. The photodissociation mass spectrum of DCF monoanion (Figure 1e, m/z 399) shows two minor fragments at m/z 355 and 354 and a major fragment at m/z 319. The former minor fragments are likely a loss of CO2 (44 Da) and COOH (45 Da) and the latter is likely losses of CO2 and HCl (44 Da plus 36 Da). Isolation of the fragments at m/z 355 and 354, formed from collision-induced dissociation of the monoanion precursor, and subsequent irradiation of those fragments (i.e., an MS3 experiment) using the same excitation wavelength and power does not yield significant further fragmentation to form m/z 319 (Figure S1 in Supporting Information). This indicates that the m/z 319 fragment is formed directly from the precursor ion, rather than being a second generation fragment formed by photodissociation of the m/z 355 and 354 fragments. The m/z 319 fragment is also observed as the major product from the IRMPD experiment.34 The photo-

avalanche diode (PDM series, Micro Photon Devices, Montreal, Canada) for fast optical detection. More details about the fluorescence lifetime setup can be found elsewhere.38 The mass spectrometer and laser excitation settings were the same as used to measure the steady-state fluorescence spectra. Each of the lifetime decays shown is an integration of 1800 onesecond acquisitions.

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RESULTS AND DISCUSSION Photodissociation. Irradiation of the monoisotopically isolated cationic, monoanionic, and dianionic charge states of DFF and DCF with visible light yields photodissociation fragments (Figure 1) similar to those observed from FL ions.33 The photodissociation mass spectra of the DFF and DCF cations are the most complex among the three charge states. Both cations show major fragments that correspond to a loss of 46 Da, same as the FL cation,33 which is likely a loss of formic acid from the pendant carboxyphenyl moiety. In addition, 1354

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Table 1. Summary of Spectral Features in the Electronic Action Spectra of Gaseous Fluoresceins Shown in Figure 2a cations FL DFF DCF a

monoanions

dianions

λmax/nm

shoulder/cm−1

fwhm/cm−1

λmax /nm

fwhm/cm−1

λmax /nm

shoulder/cm−1

fwhm/cm−1

425 (443) 425 (438) 430 (450)

+860

1400 1400 1590

525 (424, 466) 525 (450, 475) 530 (465, 490)

2600 2180 2140

500 (490) 500 (490) 505 (502)

+1280 +1280 +1030

1200 1300 1300

UV/visible absorption maxima in water are shown in parentheses for comparison.

thus, the measured absorption spectra at intermediate pH is predominantly due to the contribution of the monoanions. Because cations and dianions exist as single tautomers,34 shifts in the electronic spectra are attributed to the relative stabilizations of ground and excited electronic states provided by the surrounding water molecules. The visible absorption spectra of the cations in aqueous solution lie at lower energy than the intrinsic gas-phase values by 700−1030 cm−1. This indicates that the excited is preferentially stabilized over the ground state upon hydration. The opposite is observed for the dianions, where maxima for the aqueous FL and DFF solution spectra lie at higher energy than their gas-phase electronic action spectra by ∼370 and 410 cm−1, respectively. This blue shift is much smaller in DCF dianion, just ∼80 cm−1. Thus, it appears that solvent interaction preferentially stabilized the ground state over the excited state of the dianions. We note that the blue shift observed upon hydration is contrary to the results from calculations on fluorescein dianion at the TDDFT/BP86(6-31G**) level, which found that the aqueous maximum is red-shifted by ∼840 cm−1 from the gas phase.28 The discrepancy with the electron structure theory is likely due to the lack of diffuse functions in the basis set selected for computation; these are generally required to accurately describe anions. The direction of the shift, though not the magnitude is in agreement with computations of Silva et al., who used a sequential Monte Carlo/quantum mechanical (INDO/CIDS) methodology to simulate the effects of specific interactions with water molecules on the absorption spectrum of the fluorescein dianion.40 The opposing behavior of the cations and the dianions can be attributed to differences in the hydrogen bonding (HBD) interactions between the xanthene chromophore of the cationic and dianionic ions and the water molecules.39 Dianions form HDB interaction with water through electron lone pair on the oxygen atom (of the solute) and positively polarized hydrogen atom of water. Upon electronic excitation, the electron density around the lone pair becomes more diffuse but the presence of HBD interactions opposes this diffuseness. This results in a less polarizable excited state in a media with high HBD capacity and hence the blue shifts in water. This is termed the “reverse solvatochromic effect”. Indeed, the maximum of absorption spectrum of FL is the non-hydrogen bonding solvent dimethyl sulfoxide lies to the red of the gas-phase excitation maximum.33 The opposite occurs (ie, the normal solvatochromic effect) for the cations that form HBD interaction through positively polarized hydrogen (of the solute) and electron lone pair on the oxygen atom of water. The red-shift observed upon hydration of the fluorescein cations is similar to the behavior observed for cationic rhodamine.37 When excited, the HBD interactions facilitate the diffusion of electron density and this results in an increased polarizability of the excited state. The monoanions present by far the largest change in properties when moving from the aqueous to the gas phase. The maxima of the aqueous visible absorption spectra lie

dissociation spectrum of DFF monoanion (m/z 367) shows three major fragments that are similar in intensities at m/z 323, 322, and 303. Very similar to DCF monoanion, they are likely due to the loss of CO2, COOH, and CO2 and an additional hydrogen fluoride (44 Da plus 20 Da). Irradiation of m/z 323 or 322 formed by CID yields a minimal amount of m/z 303, again suggesting that this fragment is formed directly by photodissociation of the precursor ion. Interestingly, the major deactivation pathway in all the dianions [(M − 2H)2−] is electron photodetachment with the formation of radical monoanions [(M − 2H)•−], which appear one m/z unit lower than the even electron monoanions [(M − H)−] formed directly from the electrospray. Isolation of the radical monoanion, formed from electron photodetachment of the dianion, and subsequent irradiation using the same excitation wavelength and power yields further dissociation to form m/z 321(for DFF) and 353 (for DCF) (Figure S2 in Supporting Information). Electronic Action Spectroscopy. Figure 2 compares the electronic action spectra measured for three different charge states of gaseous FL, DFF, and DCF as well as the visible absorption spectra of those ions in aqueous solution. Spectral features are summarized in Table 1. For each charge state, the electronic action spectra of DFF and DCF are similar to those of FL in terms of spectral shapes as well as maxima. The measured maxima of the three charge states of DFF are the same as those of FL and are 5 nm to the blue of the values found for DCF. The cations lie furthest to the blue with maxima in the electronic action spectra at 425 (FL and DFF) and 430 nm (DCF). A shoulder ∼860 cm−1 higher in energy than the maximum is evident in the FL cation spectrum whereas no distinct shoulder appears in the action spectra of DFF and DCF cations in the wavelength range scanned. For the dianions, the maxima are at 500 nm (FL and DFF) and 505 nm (DCF) and a distinct shoulder is visible ∼1250 cm−1 above the maxima for all three analogues. For the monoanions, the maximum is around 525 nm for DFF and FL, whereas for DCF, the maximum again lies somewhat to the red of this value, at 530 nm. Due to the low power output from the laser above 520 nm, the electronic action spectra of the monoanions in the range of 500−540 nm were remeasured with a lower irradiation power (insets of Figure 2) to determine the spectral maximum of the DCF monoanion. The fwhm values of the cation and dianion action spectra are similar and are approximately 50% of the fwhm of the monoanion action spectra (Table 1). For comparison, Figure 2 also shows with solid lines visible absorption spectra of FL, DFF and DCF in aqueous solutions at low pH (left-hand column, predominantly cationic species in solution), high pH (right-hand column, predominantly dianionic species in solution) and an intermediate pH (middle column, a mixture of monoanions and neutrals). Experiments30 and calculations35 have shown that neutral fluorescein exists mostly as lactones in water which do not absorb visible light; 1355

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significantly higher in energy than those of the gas-phase electronic action spectra, by 4540, 3080, and 2730 cm−1 for FL, DFF, and DCF monoanions, respectively. The solution spectra of DFF and DCF monoanions is considered to have two maxima and the one at higher wavenumber was used for calculating the shifts because only the blue edges of the gasphase spectra are well-defined. These shifts are significantly larger than those observed for either the cations or the dianions. This observation suggests that the gaseous monoanions exist predominantly in different conformations from those present in aqueous solution for DFF and DCF as we have previously proposed for FL itself.33 In particular, the results of the electronic spectroscopy indicate that the chromophore (the xanthene ring) is significantly altered between solution and the gas phase. Infrared multiple photon dissociation experiments and electronic structure calculations support this conclusion and further indicate that the dominant monoanionic form in the gas phase is deprotonated on the xanthene ring system (tautomer Mono_X, Scheme 1), as compared to the deprotonation of the carboxylate on the pendant carboxy phenyl group (tautomer Mono_XH, Scheme 1) favored in aqueous solution.34 Computational results for FL and DCF have been discussed previously34 whereas those for DFF are shown in the Supporting Information (Table S3). By far the most stable conformers of the FL, DFF, and DCF monoanions are Mono_X tautomers. These are more stable than the lowestenergy Mono_XH conformers by 17−32 kJ/mol at the MP2/6311+G**//B3LYP/6-31+G* level of theory. We note that the xanthene chromophore in the Mono_X tautomer is deprotonated and closely resembles that of the dianions; thus, the similarity of the maxima in the electronic action spectra of the mono- and dianions lend strong support to this assignment. The stabilization of distinct tautomers, deprotonated either at the xanthene chromophore or at the pendant carboxy phenyl group, has also been reported for DCF monoanion in solution; in aqueous solution, the Mono_XH tautomer dominates whereas in a ternary solvent mixture of benzene-ethanol− water with tautomer Mono_X is the dominant form.22,41 Photodissociation Power Dependence. The power dependence of precursor ion depletion is shown in Figure 3. Clearly, the photostability of a given charge state of the three fluorescein analogues do not differ much from each other. In solution, the dianions of DCF and DFF are significantly more stable against photobleaching than FL itself;9,13 however, this is not reflected in the gas-phase photodissociation data of the dianions. Whereas DCF requires the most energy to photofragment in the gas phase, DFF is dissociated more readily than FL itself. Of all three charge states, cations require the highest power to dissociate (Figure 3a) and show the most complex dissociation pattern with multiple accessible dissociation channels clearly present (Figure 2). The natural logarithms of the remaining precursor ion fraction can be fit with a fourthorder polynomial dependence on power (Figure 3a), which indicates that the absorption of multiple photons is responsible for the photodissociation of the cations. Moreover, the fragments formed closely resemble those from the infrared multiple photon dissociation (IRMPD) study.34 Taken together, this information strongly suggests that the visible photodissociation of the cations (and the monoanions) occurs on the ground electronic surface. This has been explained by invoking a mechanism37 similar to that of IRMPD.42,43 Briefly,

Figure 3. Power dependence for dissociation of FL, DCF, and DFF (a) cations, (b) monoanions, and (c) dianions measured at excitation wavelength 430 nm (cations), 525 nm (monoanions), and 500 nm (dianions). Power dependence measurements are shown as natural logarithm of fraction of remaining precursor ion. The plots were constructed by removal of any nondissociating component (see text). Dotted, solid, and dashed lines show polynomial fits to FL, DCF, and DFF data, respectively.

after the cation absorbs a visible photon and gets elevated to the excited electronic state, it can undergo internal conversion to return to some excited vibrational level of the ground electronic state. Intramolecular vibrational energy redistribution (IVR) redistributes this energy throughout the many vibrational modes of the molecule, the ground-state cation can now reabsorb another visible photon, and the cycle repeats. The requirement for high dissociation power must be the result of a combination of rapid IVR and high dissociation threshold energy. The observation of multiple dissociation pathways suggests that many bonds within the cations have similar dissociation energies. Photodissociation of the monoanions also exhibits a complex power dependence. One possible reason for a complex power dependence is the presence of multiple conformational populations. Though electronic structure calculations find multiple conformers, the most stable of these are all of the Mono_X form, which have virtually the same conformation of 1356

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rescence from the FL dianion is attributed to the ease of the electron photodetachment de-excitation pathway,33 which apparently out-competes fluorescence as a deactivation pathway in the gas-phase dianions. The same is likely to be true for both DFF and DCF dianions, which show electron detachment cross sections similar to that observed for the FL dianion. The lack of fluorescence from the cations is also not surprising. In solution, the cations are believed to undergo two excited-state proton transfers to solvent to form FL monoanions, which then emit fluorescence.24 In the gas phase, there are no neighboring solvent molecules to accept such a proton transfer, thus eliminating this de-excitation pathway. Interestingly, we are able to detect fluorescence signal from the monoanions of all three dyes (Figure 4) although the

the xanthene chromophore and differ only by the conformation of the pendant carboxy phenyl group. Mono_XH tautomers lie significantly higher in energy (17−32 kJ/mol), thus suggesting that there is only one major population.34 Moreover, we note that photodissociation kinetics of FL, DFF, and DCF monoanions are well fit by single exponentials (Figure S4 in Supporting Information), again suggesting that there is a single photodissociating population of the monoanions. The natural logarithms of the precursor ion fractions show second-order polynomial dependence on power (Figure 3b), which when combined with the assumption of a single population indicates that the absorption of multiple photons is responsible for the photodissociations of the monoanions. We also note that small amount of nondissociating component, 6% in Fl, 3% in DFF, and 9% in DCF, was observed. This nondissociating component was subtracted in the analysis shown in Figure 3. The unsubtracted data may be seen in the Supporting Information. The nondissociating component may be the result of an isobaric chemical contaminant or due to the presence of a small portion of a “dark state”, a species that does not absorb light at the excitation wavelength used. The latter could a lactone or a long-lived triplet state. We note that neutral FL lactone is well-known.35,44,45 Electronic structure calculations of the gas-phase monoanions indicate that gaseous monoanionic lactone conformers are significantly higher in energy (21.7 and 16.7 kJ/mol in FL and DCF, respectively) than the Mono_X global minimum,34 making their presence unlikely. Furthermore, photodissociation of the chlorinated (A +2) isotopic peak of DCF yields almost complete depletion of the precursor ion suggesting that the cause of the nondissociating component is mostly likely a chemical contaminant. The dianions show the simplest dissociation pattern among the three charge states. Significantly less energy is required to cause the dianions to undergo electron photodetachment than to fragment the monoanions and the cations. Photodissociation mass spectra of all three dianions show a small amount (less than 5%) of nondissociating species. The amount of nondissociating component changes from day to day and hence is likely due to the presence of an isobaric chemical contaminant. Upon removal of these nondissociating components, the natural logarithm of precursor ion fraction decreases linearly with power, as shown in Figure 3c. The power dependence measurements of the dianions thus suggest that electron photodetachment of the dianions, the observed dissociation pathway, is a single-photon process. The effective electron detachment cross sections, obtained from the negative slopes of the natural logarithm plots, are 3.96 Å2 for FL and 4.14 Å2 for DFF and DCF dianions. The similarity of these values indicates that the substitution of halogen atoms into the 2′ and 7′ positions of the xanthene ring only has a very small effect on the likelihood of electron detachment process. The simple power dependence and first-order dissociation kinetics (not shown) agree with the results of IRMPD action spectroscopy and electronic structure calculations, which also indicate the presence of a single conformer of the dianions in the gas phase.34 Fluorescence Emission Spectroscopy. FL and its derivatives are among the most common fluorescent dyes. In solution they emit bright fluorescence, with the quantum yield of the fluorescein dianions of ∼0.9. However, in the gas phase the dianionic and cationic forms of FL,33 DFF and DCF do not emit detectable fluorescence. The lack of detectable fluo-

Figure 4. Comparison of fluorescence emission spectra of FL (blue), DFF (red), and DCF (purple) monoanions and rhodamine 640 (gray). Smoothed spectra, obtained using the Savizky−Golay method with a second-order polynomial and 90-point windows, are shown in solid lines of the same colors. Emission spectra are measured with 510 nm excitation light using identical numbers of ions, irradiation power and time for each species.

intensities of the fluorescence emission are low, less than 50% of that emitted by the same number of rhodamine 640 ions measured under the same experimental conditions. The excitation efficiency of rhodamine 640 is less than 20% at the wavelength used46 whereas this wavelength is much nearer the excitation maxima of the fluorescein derivatives (Table 1). Thus, the brightness of the three monoanions is less than 10% that of rhodamine 640. Among the three monoanions, the fluorescence intensity of the FL monoanion is the lowest, about 15% less bright than that of DFF and DCF monoanions. Furthermore, to compare the relative brightness of fluorescein analogues at their respective excitation maxima, the emission of FL, DFF, and DCF monoanions needs to be scaled up by a factor of ∼1.2, 1.4, and 1.6 respectively. These numbers are obtained by comparing the amount of photodissociation at the excitation wavelength (510 nm) and the respective excitation maxima (Figure 2). This scaling further increases the difference among the relative brightness of the dyes. Thus, the relative brightness of the gaseous monoanions follows the order FL < DFF < DCF. 1357

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explanation is that an ensemble of different forms of gas-phase monoanionic dyes exists. These could be different tautomers corresponding to different deprotonation sites in the absence of solvent molecules and/or distinct rotamers. However, photodissociation kinetics of the monoanion suggest the presence of a single absorbing population and computed low energy conformers differ only in the pendant group, rather than the xanthene chromophore.34 Broad emission spectra may also result when the excited-state lifetime is very short.56,57 This possibility is ruled out as the lifetimes of the monoanions are measured (Figure S5 in Supporting Information) to be ∼5 ns, similar to the lifetimes of gaseous rhodamine dyes.38 Thus, the underlying cause for the broad emission profile of the fluorescein monoanions is at present unclear.

It is interesting but not surprising that DFF and DCF monoanions emit brighter fluorescence in the gas phase than the FL monoanions, as they were synthesized to be superior alternatives to FL. In basic solution, DFF and DCF exist as almost 100% dianions and have reported quantum yields of 0.9728 and 0.94,29 respectively. Even though these values are higher than the 0.90 for FL,7 the difference in solution quantum yields of the dianions alone is insufficient to explain the difference in the gas-phase fluorescence intensities of the monoanions. It also does not predict the observed trend in the relative brightness of DFF and DCF: DCF, whose dianion has a slightly lower quantum yield in solution than DFF, has a brighter monoanion in the gas phase. Here, we would like to discuss two possible competing deactivation pathways that could affect the relative brightness of the monoanions of the two fluorescein analogues: photoinduced electron transfer (PET) and intersystem crossing. PET is a well-known competitive decay pathway in dianionic fluoresceins in solution.47−49 In the fluorescein dianion, the pendant benzene moiety serves as an electron donor to the excited three-ring xanthene chromophore, which is the electron acceptor. The likelihood of the PET process can be assessed by evaluating the oxidation (of the benzoate) and reduction (of the excited xanthene) potentials. Zhang and co-workers studied aqueous fluorescein dianions with di- and tetrachlorinated xanthene and reported less negative reduction potentials of the chlorinated xanthene and smaller rate constants for the PET process compared to that of FL itself.50 Hence, longer fluorescence lifetimes and higher quantum yields of the chlorinated fluoresceins result. A similar explanation may be put forth for the monoanions investigated here: halogenation of the xanthene ring decreases the rate of electron transfer to the excited xanthene ring, thus decreasing PET and increasing quantum yield of the monoanions. Another factor that affects quantum yield, and may therefore affect brightness, is the rate of intersystem crossing. The heavy atom effect suggests that the incorporation of heavy atoms such as Cl, Br, and I generally enhances the rate of intersystem crossing,51,52 thus increasing the triplet quantum yield and decreasing the fluorescence quantum yields of many dyes.53−55 The observed order in brightness: DCF > DFF > FL, cannot be explained by the heavy atom effect, which would predict the opposite order. The lack of a significant heavy atom effect in chlorinated fluoresceins has been observed previously in solution. Zhang and co-worker report that for chlorinated fluoresceins in aqueous solutions, chlorination decreases the rate of intersystem crossing compared to that in FL itself.50 On the other hand, bromination and iodination seem to have the normal heavy atom effect on fluoresceins in aqueous solutions.50 Finally, we note that the gas-phase fluorescence emission spectra of the monoanions of Fl, DFF, and DCF are not mirror images of the action spectra, unlike observations for other gaseous xanthene dyes.37 In fact, the emission spectra appear to be unusually broad and unconstructed. There are several possible explanations. There could be a large geometry change between the ground (S0), and the first excited electronic (S1) states, resulting in a large Franck−Condon envelope and broadened excitation and emission spectra. A large change in geometry between ground and first excited electronic states is also consistent with the significantly larger Stokes shifts (∼1650 cm−1) of the monoanions compared to the values reported for several rhodamine dyes (∼500 cm−1).37 A second possible

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CONCLUSIONS In summary, the gas-phase electronic action spectra of DFF and DCF cations, dianions, and monoanions were found to be similar to those of FL ions. The maxima of the three charge states of DCF are red-shifted relative to the respective FL and DFF ions, which agrees with the trends observed from solution absorption spectra. The dianions of all analogues undergo electron photodetachments upon absorption of a single visible photon. The power dependence and dissociation pathway of the cations and monoanions are more complex. Weak fluorescence was observed from monoanions of all species, whereas no fluorescence was detected for the cations and the dianions. Studies of gaseous ions such as the one reported here provide insight into the effect of solvent interactions on the photophysical properties of those dyes by comparing their intrinsic properties measured in vacuo with previous studies done in solutions. In this work, it has enabled elucidation of the effects of substitution on the intrinsic fluorescent properties of the dyes and separation of this from solvent effects. In the future, these findings can be used to develop intrinsically more robust and efficient dyes.

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ASSOCIATED CONTENT

S Supporting Information *

Photodissociation mass spectra of the radical monoanions, photodissociation mass spectra of major product ions, polynomical fits to photodissociation power dependence measurements, photodissociation kinetics of monoanions, fluorescence lifetime measurements of monoanions, and computed structure and relative free energies of DFF dianions, monoanions, and cations. 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]. Tel. +1 416-946-7198. Fax: +1 416-978-8775. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors gratefully acknowledge support for this work provided by the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council of Canada, the Canada Research Chairs Program, and the Province of Ontario. 1358

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