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Gas-Phase Ion Spectroscopy of Congo Red Dianions and Their Complexes with Betaine Christina Kjær, James M. Lisy, and Steen Brøndsted Nielsen J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b00904 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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The Journal of Physical Chemistry

Gas-Phase Ion Spectroscopy of Congo Red Dianions and Their Complexes with Betaine Christina Kjær,a James M. Lisy,b and Steen Brøndsted Nielsena,* a

Department of Physics and Astronomy, Aarhus University, Denmark

b

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

ABSTRACT Congo Red (CR) is an azo dye that is negatively charged in aqueous solutions. Here we report on the intrinsic electronic properties of CR dianions from mass spectroscopy experiments on bare dianions and their complexes with betaine (B). As betaine is a zwitterion, it possesses a large dipole moment and is a good reporter on the sensitivity of CR to microenvironmental changes. Photoexcitation of CR2- in the visible region resulted in several fragment ions after absorption of at least three photons, with major fragmentation routes due to breakage of one or both C–NN bonds, one azo linkage, and/or the bonds to sulfite. Their yields as function of excitation wavelength reveal a broad absorption in the visible region with the lowest-energy band located at ~500 nm. Features are observed with a spacing of ~1500 cm-1. One photon was sufficient to dissociate CR2-•B, and its action spectrum was almost identical to those of CR2- in accordance with previous findings that a symmetric ion is essentially unaffected by changes in its microenvironment. Electron detachment occurs in the UV with threshold energy of 3.6±0.1 eV for CR2- and 3.81±0.06 eV for CR2-•B. Attempts to measure fluorescence from photoexcited CR2- were unsuccessful.

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INTRODUCTION Congo Red (CR) is an azo dye with a symmetric structure and carries two negative charges after deprotonation of its two sulfonic acid groups (Figure 1). The azobenzene motif is often employed in complex photoswitches as trans-to-cis isomerization is induced by UV-light.1 As CR2- contains two azo groups (–N=N–), there are three possible isomers: EE, EZ (equal to ZE as the molecule is symmetric), and ZZ where the EE geometry dominates in solution. The absorption is in the visible region due to the extended π-conjugated network that involves all the aromatic rings and the azo groups and is ππ* in character.2 Photoisomerization to EZ (or ZE) is fast, within picoseconds, which makes the molecule an attractive photoswitch.3 In solution, CR is only weakly fluorescent with quantum yields of less than one percent.3,4 In biology, CR is used as a staining agent,5-7 e.g., as an amyloid marker in detection of amyloid fibrils connected to Alzheimer diseases.8 Its binding affinity is particularly high to αhelical structures.9 CR can self-assemble to form supramolecular structures that, different from individual CR molecules, target proteins.10 Finally, it serves as a pH indicator with its color bright red in solutions with pH > 5, while changing towards the blue for pH < 3.2,3,11 The spectroscopic properties of dyes often depend on their immediate environment, e.g., solvent molecules or counter ions, a fact exploited extensively to report on microenvironments in biological systems from absorption or emission band maxima.12 In recent years significant work has been done in elucidating the intrinsic properties of ionic dyes, some acting as photoswitches like the azo dyes, from spectroscopy experiments on isolated ions in vacuo.13-22 For example, protonated azobenzene and other photoswitches were studied by Bieske and co-workers20-22


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using photoisomerization action spectroscopy (PISA). These experiments take advantage of different ion mobilities of the two isomers when they travel through a drift tube filled with N2 as their collision cross sections differ. In other experiments, Jockusch and co-workers13 measured the fluorescence from photoexcited rhodamine dye ions stored in a quadrupole ion trap to uncover the effect of solvation on the emission properties of the ions. One driving force for gasphase spectroscopy is that a firm understanding of the spectroscopic properties of the bare ions is relevant to decipher the influence of different solvents or biological environments. In this work, we shed light on the intrinsic spectroscopic properties of CR dianions (CR2-) when isolated in vacuo based on gas-phase ion spectroscopy, that is, in the absence of a microenvironmental influence. Experiments were also performed on complexes between CR2- and betaine (B), as such complexes are easily photodissociated in contrast to the bare ions (vide infra). Betaine is a zwitterion containing both a quaternary ammonium ion and carboxylate group, and as a result possesses a large dipole moment (11.9 Debye23). The binding to a negatively charged ion such as CR2- is due to the ion-dipole interaction, typically with a binding energy of about 1 eV.24 Despite the potential effects due to the large electric field, betaine was earlier shown to only weakly modify electronic transition energies of symmetric ions.24 In contrast, anions that undergo charge-transfer transitions are strongly affected by the presence of a large electric dipole (their absorption is shifted to shorter wavelengths as charge density moves away from the dipole). Spectroscopy of complexes with betaine, readily formed by electrospray ionization, is an easy way to obtain gas-phase action spectra where dissociation is the result of the absorption of a single visible or UV photon. This approach circumvents the complication of interpreting the photofragmentation spectrum of the bare dianion, resulting from multi-photon absorptions.


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Indeed, large ions, with many degrees of freedom or for which the barriers for dissociation are high, often do not dissociate within the instrumental time window after the absorption of a single photon.

EXPERIMENTAL SECTION Action spectroscopy: Gas-phase spectroscopy experiments were carried out using a home-built accelerator mass spectrometer (sector instrument) equipped with an electrospray ion source and a tunable laser system. The instrument is described in detail elsewhere.25,26 Methanol solutions of either CR or CR with betaine were electrosprayed. The spray solution was made by dissolving a few grains of CR in 1 mL methanol. A few grains of betaine were added to the solution to make complexes. We estimate the concentrations to be about 10 mM. No difficulties in producing ions from day to day were encountered, suggesting slight variations in concentration were unimportant. All ions were accumulated in an octopole ion trap that was emptied every 25 ms (40-Hz repetition rate) to produce an ion bunch. After acceleration to 50 keV (times the charge state) kinetic energy, ions of interest were selected based on their mass-to-charge ratio using a bending magnet (m/z 325 for CR2- and m/z 383.5 for CR2-•B; mass of betaine is 117). These were then irradiated by light from a pulsed tunable laser system (EKSPLA model NT 342B-SH-20). The fundamental (1064 nm) from a Nd:YAG laser was frequency tripled to 355 nm in the UV to pump an optical parametric oscillator, generating a visible (420-720 nm range) and an infrared idler photon. Tunable UV photons (210-419 nm) were produced by frequency doubling the visible photons in a barium borate crystal. The laser was operated at a 20-Hz repetition rate, i.e., irradiating every other ion bunch to correct for background fragmentation. Fragment ions were


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analyzed by a hemispherical electrostatic analyzer (ESA) according to their kinetic energy per charge and then amplified by a channeltron detector for pulse-counting detection. The photoinduced signal was obtained as the difference between the signal from the irradiated and nonirradiated ion bunches. Solution-phase absorption spectroscopy: The absorption spectrum of CR2- in an alkaline aqueous solution (NaOH(aq), pH 10) was obtained using an Evolution 300 Thermo spectrometer. The concentration of CR was 1.6 · 10-4 M and the cell length 100 µm. Luminescence spectroscopy: The setup for gas-phase luminescence experiments called LUNA (LUminescence iNstrument in Aarhus, also described previously27) utilizes a cylindrical Paul trap joined with an optical spectrometer. Ions were again produced by electrospray ionization and accumulated in an octopole trap. Every 50 ms the ions were transferred to the Paul trap and translationally cooled to the center of the trap from collisions with helium buffer gas. The ions were trapped by RF and DC voltages on the cylinder electrode that also facilitated mass selection. Mass-selected ions were photo-excited by the same 20-Hz repetition rate laser system described above, and emitted photons were collected and detected by an ANDOR spectrometer and CCD camera. The experiment was repeated in alternating cycles of 100 measurements with ions in the trap and 100 with an empty trap in order to correct for scattered light. Experiments were carried out with three excitation wavelengths: 488 nm, 513 nm, and 560 nm.

RESULTS AND DISCUSSION Photoinduced dissociation (PID) mass spectra: There are several fragmentation channels for CR2- after photoexcitation in the visible region (Figure 2A where λ = 470 nm) while only one


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channel was observed for the betaine complex, loss of betaine (Figure 2B where λ = 455 nm). Due to saturation of the detector, the mass regions close to the parent ions were not measured; these are indicated by grey bars in the figure. The relative importance of each fragment ion formed from the bare ions is summarized in Table 1, and the bonds that are broken are indicated in Figure 3. Experiments were also done on m/z-326 parent ions (one 34S or two 13C) to verify the number of sulfur atoms in the daughter-ion assignments. The m/z-284 peak was substantially narrower than the other fragment mass peaks and can be explained as follows. The peak width originates from the initial spread in velocities of the parent ions after acceleration to 100 keV and the kinetic energy released (KER) when the parent ion dissociates. The ESA analyzes for kinetic energy per charge so the peak width associated with a doubly charged daughter ion is half that of a singly charged with the same m/z when the KER is the same. Based on this analysis, the m/z284 peak was assigned as a fragment dianion. It should be noted that the relative fragment ion yield cannot be deduced from the spectrum in Figure 2A that took about an hour to record, as corrections for ion-beam variations in the PID mass scan are not possible over that time period; the relative importance was established in separate, independent measurements, where ion abundances were measured three separate times and averaged. The mass resolution of the mass-analyzed ion-kinetic-energy scan is approximately +/- 1 mass unit, thus mass assignments could be off by 1 amu, and we can therefore not exclude H-atom transfer between two fragments. This uncertainty in mass implies that the red lines in Figure 3 that indicate the bonds that are broken do not exclude rearrangement involving the movement of a H-atom during fragmentation. The most prominent fragment ion from bare CR2- was m/z 414, and in Figure 3 we give two possible assignments. One is simply based on breaking one azo linkage, while the other corresponds to breaking one C-NN bond


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together with loss of NH2. The m/z-414 daughter ion resulted from the absorption of more than one photon, as evidenced from the yield versus relative laser power at 455-nm excitation (Figure 4A, maximum laser power corresponds to 1 while 0 is with no laser). A fit to the function, f(x) = axb, shows that on average three photons (the b exponent) are needed to form this fragment ion within the time window for dissociation (up to 10 µs). Data points above a relative laser power of 0.3 were not included in the fit due to saturation (all ions excited in interaction region). A similar dependency on the laser power was found for the other fragment ions with the exception of the smallest (m/z 64) that required one additional photon. This is illustrated for four of the fragment ions by their almost constant ratios relative to the m/z-414 fragment ion in Figure 4B. Results for all fragment ions can be found in Supporting Information. In contrast, the yield of CR2- from CR2-•B increased linearly with laser power until saturation was reached (see Figure 4C), which implies a one-photon dissociation process. Excitation in the UV results in electron detachment for both CR2- and CR2-•B to give CR-• (m/z 650) and CR-••B (m/z 767), respectively (Figure 5A and 5B). This channel is clearly dominant for the bare dianions, while loss of betaine remains the main channel for CR2-•B to give CR2- (m/z 325) (Figure 5B) even though the photon energy is well above the detachment energy (vide infra). Internal conversion can therefore compete with coupling to the continuum. Electron detachment together with loss of betaine is also detected through the observation of CR•

.

Action spectra: First, we present the results for the bare CR dianions in the visible region. Figure 6A shows the difference between ‘laser-on’ and ‘laser-off’ signals for the formation of the


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most abundant fragment ion (m/z 414) versus excitation wavelength. It is the sum of 11 scans. The ‘laser-off’ signal is a result of the decay of metastable parent ions or dissociation after collisions between parent ions and residual gas in the beam line. To obtain the action spectrum, the PID signal is divided by the number of photons (in the laser pulse) cubed (see Figure 6B). A 10-point sliding-average line is included to guide the eye. The grey area depicts the variation in the spectra if the formation was a result of absorption of two or four photons, respectively. As it is difficult to firmly establish the number of photons absorbed when both a non-linear power dependence and saturation (at high laser powers) are present, this additional analysis is presented to show the impact arising from the uncertainty in the number of photons absorbed. Fortunately, the band maxima are only weakly dependent on the correction for the number of photons as there is little variation in laser power across this region where the absorption is strong. An exception is at the short wavelength limit of the spectral region where the laser power changes significantly; as a result, we only show results down to 428 nm. The action spectra for the formation of the remaining fragment ions were obtained in the same manner (sum of three scans), and all look similar, which simplifies the analysis (see Figure 7A). The individual action spectra were combined according to their importance at 455 nm to give the total action spectrum of CR2(Figure 7C).28 In the case of CR2-•B, the yield of CR2- was divided by number of photons in the laser pulse to obtain the action spectrum (sum of six scans, Figure 7B). The spectra of CR2- and CR2-•B are quite similar (see Figure 7C): Both ions exhibit absorption from 430 nm to 570 nm, a double band structure and indications of a third shoulder. While the spectral resolution is not very high, the double band structure was evident in individual scans. Also the similarity between the spectra indicate that the double band structure cannot be the result of the correction for the number of photons absorbed, which while nontrivial for the


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bare ion case, is simple in the single photon case for the complex. The vibronic bands are separated by approximately 1500 cm-1, possibly associated with skeleton stretch modes. Indeed, the Raman spectrum of the disodium salt of CR obtained at 1064-nm excitation displays bands at 1156 cm-1 (phenyl–N stretching mode), 1376 cm-1 (napthyl ring C–C stretching mode, 1401, 1451 cm-1 (–N=N– stretching), and 1592 cm-1 (phenyl ring C–C stretching mode) as recently reported by Costa et al.3 However, as we cannot separate different isomers in our experiment, it is also possible that the band structure is associated with two or three isomers in the ion beam (EE, ZE, and/or ZZ). To firmly address this would require ion-mobility experiments and photoexcitation of individually selected isomers as done by Bieske’s group. It is noteworthy though that if the absorption spectrum is the result of more than one isomer, betaine attachment does not significantly alter the isomer distribution. The absorption by CR2- in alkaline water (pH 10) is also included in Figure 7C and reveals a maximum at 492 ± 4 nm. The band maximum is similar to that of CR in water (499 nm) and aqueous Na2SO4 solution (494 nm) but much further to the blue than that in DMSO solutions (534 nm) with values reported by Yamaki and co-workers11. As in the gas phase, our solution-phase spectrum is broad but it extends to even longer wavelengths (up to about 600 nm). The band maximum coincides with that of the lowest-energy transition in the gas phase but the higher-energy features do not stand out in the solution-phase spectrum. The gas-phase experiment tend to favor higher photon energies, as we rely on dissociation within a few microseconds. This could skew the spectrum somewhat, and this may explain that the gas-phase absorption appears higher than in solution-phase at wavelengths shorter than 470 nm. The blueshift seen between water and DMSO has earlier been explained based on H-bond interactions between the protic solvent with the amino and/or azo groups of the dye.11 Such an


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explanation, however, seems inconsistent with the close similarity between the gas-phase spectrum and the water spectrum. If anything, it appears that water molecules somewhat shift the overall band to longer wavelengths. An nπ* transition involving the lone pair electrons of the amino group would expectedly shift to shorter wavelengths when a water molecule hydrogen bonds to the nitrogen. The shift in absorption in DMSO compared to the gas phase is likely due to the ππ* character of the electronic transition; however the origin of the large difference between water and DMSO remains unclear (unless dimerization occurs). The action spectrum for the formation of CR2- from CR2-•B is shown in Figure 8A in the wavelength region from 210 nm to 415 nm. A linear dependence on the photon energy is assumed as the photon energies are higher in the UV than in the visible region. Absorption is observed over the entire region although it is minor above 400 nm, with the most intense features seen at about 315 nm and 350 nm. Notice that above 355 nm the laser power is much lower than that in the lower wavelength region due to the inefficient frequency doubling of the idler output. Again, absorption occurs over the same region as in aqueous solution, which is also given in Figure 8A. In solution, there is a strong band with maxium at about 340 nm, and the transition is suggested to involve the NH2 group.2 A direct comparison between gas phase and solution phase is not simple, and proper band assignments will await more experimental work where the number of isomers present in the gas-phase experiment can be established. A similar gas-phase experiment was not possible for CR2- as the laser power was too low to cause enough photodissociation. To establish the threshold electron detachment energies of CR2- and CR2-•B, the yields of m/z-650 and m/z-767 ions, respectively, were measured as a function of wavelength (Figure 9A


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and 9B). We are not aware of a threshold law that applies for dianions, so for simplicity we performed linear fits to the data. The threshold energy is at 3.6 ± 0.1 eV (345 ± 10 nm) for CR2and 3.81 ± 0.06 eV (325 ± 5 nm) for CR2-•B, where the attachment of betaine leads to an increase in the detachment energy by 0.16 eV. This is not surprising as the betaine reduces the Coulomb repulsion between the two negatively charged ends and thereby stabilizes the dianion. So the increase in the threshold energy for electron detachment from CR2-•B is expected. According to a geometry optimization of the bare dianion in EE geometry at the B3LYP/3-21G level of theory, the largest distance between the two sulfurs is 18.2 Å, which would yield a maximum Coulomb repulsion energy of about 0.8 eV. Betaine does not eliminate this repulsion, but provides a reduction of about 20%. In previous work, the binding energy of betaine to different monoanions was calculated to be about 1 eV.24 Therefore to detach the electron from the sulfite bound to betaine would give an increase in detachment energy of about 1 eV assuming that the interaction energy between neutral SO3 and betaine is zero. The measured increase is well below this number and is indicative of electron detachment occurring from the free sulfite. According to the solution-phase spectrum (Figure 8A), there are electronically excited states in the region where electron detachment occurs. This increases the electron detachment cross sections for the bare ions as these excited states are unbound. The spectrum associated with formation of CR-• (m/z 650) from CR2-•B after loss of both betaine and an electron follows the spectrum associated with only electron detachment (m/z 767) but the data quality is not good enough to extract an appearance energy. Fluorescence. Experiments were first done on resorufin anions that are known to fluoresce in the gas phase29 in order to have optimum conditions for CR2-. Importantly, luminescence experiments were done with low laser power, that is, under conditions associated with single-


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photon absorption. This was to insure that photodissociation of CR2- will not occur, as the absorption of at least three photons is needed, and the ions are cooled in buffer gas collisions within hundreds of nanoseconds. However, no fluorescence was detected from CR2- following photoexcitation at 488 nm, 513 nm, or 560 nm. A gas-phase fluorescence quantum yield below the detection limit of our experiment is in accordance with the very low fluorescence quantum yield found for aqueous solutions.

CONCLUSIONS In conclusion, we have found that Congo Red dianions are not easily photodissociated in the visible region of their absorption. This implies that the action spectrum, taken to represent the absorption by the ions, is a result of a three-photon absorption process. Here the PISA approach would be useful, as only one photon is needed for isomerization to occur (assuming that the isomerization quantum yield only weakly depends on excitation energy, and that the excitation involves the N=N bridge). Complexes between CR2- and betaine are easily formed by electrospray ionization. These are weakly bound (about 1 eV), and we find that one photon is sufficient to dissociate the complex on the microsecond time scale of our experiments. The action spectra of the bare ions and the complexes are quite similar, further supporting the approach that the absorption by large and symmetric ions, that do not easily dissociate, is most easily measured from photodissociation of their complexes with betaine. The spectra are broad, which is also the case for the dianion in aqueous solution. Features are observed in the gas-phase spectra that are either due to vibronic structure or the presence of two or three isomers in the ion beam. From wavelength scans in the ultraviolet, we determined the thresholds for electron


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photodetachment. The value is higher for the complex than that for the bare ion as the betaine stabilizes the dianion. We were unable to measure any fluorescence from the bare dianions.

Table 1. Relative importance (RI) of fragment ions formed after 455-nm photoexcitation of CR2.

m/z

64

80

152

169

221

233

284

401

414

RI

0.13

0.48

0.69

0.29

0.42

0.50

0.06

0.58

1


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Figure 1. Structures of Congo Red dianions (CR2-) and betaine (B).


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Figure 2. PID mass spectra of (A) CR2- (m/z 325) with excitation wavelength 470 nm and (B) CR2-•B (m/z 383.5) with excitation wavelength 455 nm. The grey areas were not scanned due to strong signal from the parent ion resulting in detector saturation. The stars mark artifact peaks due to deflection of ions that hit the outer plate of the electrostatic analyzer. The intensity of the artifact signal is only 10% of that of the m/z-325 ion but appears higher due to saturation of the m/z-325 signal in the present spectrum.


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Figure 3. Bonds that break after photoexcitation of CR2- are indicated by the red lines (except for SO3 and SO2 fragments). The prominent fragments from the PID mass spectra are assigned as noted.


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Figure 4. (A) PID signal as a function of relative laser power for the most important fragment ion (m/z 414) from CR2-. A power fit to f(x) = axb gives b = 2.9. The last three data points are not included in the fit due to saturation. (B) The PID signals of the most abundant fragment ions after photoexcitation of CR2- relative to the PID signal of the m/z-414 fragment ion. (C) PID signal of the m/z-325 fragment ion (loss of betaine) from CR2-•B. A linear fit accounts well for the low laser power data while saturation occurs for higher powers. The excitation wavelength was 455 nm in all cases.


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Figure 5. UV PID mass spectra of (A) CR2- (m/z 325) and (B) CR2-•B (m/z 383.5). The excitation wavelength was 240 nm. The grey areas were not scanned due to strong signal from the parent ion resulting in detector saturation. The stars mark artifact peaks due to deflection of ions that hit the outer plate of the electrostatic analyzer. In (A) the intensity of the artifact signal is approximately 10 % of that of the m/z-650 ion.


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Figure 6. (A) The difference in laser-on and laser-off signal for the m/z-414 fragment ion from CR2- as a function of wavelength. (B) The action spectrum for the formation of the m/z-414 fragment ion based on a three-photon correction (black line: a 10-point sliding-average to guide the eye). An analysis based on two- or four-photon corrections give data points within the indicated grey area. (C) Laser power as a function of wavelength.


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Figure 7. (A) Action spectra for eight fragment ions formed after photoexcitation of CR2-. (B) Action spectrum for loss of betaine from CR2-•B. (C) Total action spectrum for CR2- obtained after summing the yields from all dissociation channels (black) compared to action spectrum for CR2-•B (blue). The spectrum of CR2- in aqueous NaOH solution (pH = 10) is included for comparison; absorption band maximum is at 492 ± 4 nm. The solid lines are all 10-point slidingaverages of the data.


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Figure 8. (A) Action spectrum for the loss of betaine from CR2-•B (m/z 383.5). The line is a three-point sliding-average. The spectrum of CR2- in aqueous NaOH solution (pH = 10) is included for comparison. (B) The laser power as a function of wavelength.


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Figure 9. (A) Action spectrum for photoelectron detachment from CR2- . The curves are obtained from five-point sliding-averages. The inset shows data from narrow scans limited to the region 310-370 nm. A linear fit to the data yields a threshold energy of 3.6 ± 0.1 eV (345 ± 10 nm). (B) Action spectrum for the electron detachment (m/z 767) and electron detachment plus loss of betaine (m/z 650) from CR2-•B. The inset shows data from narrow scans for the m/z-767 daughter ion limited to the region 270-330 nm. A linear fit to the data yields a threshold energy of 3.81 ± 0.06 eV (325 ± 5 nm).


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ASSOCIATED CONTENT Supporting Information. Power-dependence data for all fragment ions from CR2-. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT SBN acknowledges support from the Danish Council for Independent Research (4181-00048B) and Aarhus University Research Foundation for a Guest Professorship to JML. JML also acknowledges that this material is based on work done while serving at the U.S. National Science Foundation.

ABBREVIATIONS B, Betaine; CR, Congo Red; PID, Photoinduced Dissociation; UV, Ultraviolet. REFERENCES (1) Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc.


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