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High-cooling-efficiency cryogenic quadrupole ion trap and UV-UV hole burning spectroscopy of protonated tyrosine
Journal of Molecular Spectroscopy xxx (2016) xxx–xxx
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High-cooling-efficiency cryogenic quadrupole ion trap and UV-UV hole burning spectroscopy of protonated tyrosine Shun-ichi Ishiuchi ⇑, Hiromichi Wako, Daichi Kato, Masaaki Fujii ⇑ Laboratory for Chemistry and Life Science, Institute of Innovation Research, Tokyo Institute of Technology, 4259, Nagatsuta-cho, Midori-ku, Yokohama, Japan
a r t i c l e
i n f o
Article history: Received 27 September 2016 In revised form 12 October 2016 Accepted 17 October 2016 Available online xxxx Keywords: Cold ion spectroscopy Electro spray ionization Cryogenic ion trap Gas phase spectroscopy of biomolecules Double resonance laser spectroscopy
a b s t r a c t The cooling efficiency of a cryogenic three-dimensional quadrupole ion trap (QIT) is drastically improved by using copper electrodes instead of conventional stainless-steel ones. The temperature of trapped ions (protonated tyrosine TyrH+) was estimated based on the ultraviolet (UV) photo-dissociation spectra. The UV spectrum of TryH+ shows almost no hot bands, and thus the high cooling efficiency of the copper ion trap was proven. The temperature was also estimated by simulating the observed band contour in the UV spectra, which is determined by the population in the rotationally excited levels. From the simulations, the temperature of TryH+ was estimated to be 13 K, while that in the stainless-steel QIT was 45–50 K. In addition, to demonstrate the advantage of the copper QIT, UV-UV hole burning (HB) spectra, i.e. conformation-selected UV spectra, were measured. It was confirmed that four different conformers, AD, coexist in the ultra-cold protonated tyrosine. By comparing with the calculated Franck-Condon spectra, their structural assignments were discussed, including the orientation of the OH group. Ó 2016 Published by Elsevier Inc.
1. Introduction The combination of electrospray ionization (ESI) and a cryogenic ion trap enables us to apply high-resolution laser spectroscopy to non-volatile and large molecular ions, such as biomolecules, and supra molecular systems [1]. This methodology can provide precise conformational separation and structural determination, and their possibilities are further enhanced by combining with ion mobility spectrometry (IMS) [2]. The key technique of this methodology is buffer gas cooling in the cryogenic ion trap, which is crucial to reduce population of higher-energy conformers. The cooling process of the ions consists of two steps. At first, the injected buffer gas is cooled down by thermal contact with a cryogenic ion trap; then, trapped ions are cooled down by collisions with the cold buffer gas. Currently, two types of ion traps are used for this purpose, mainly: linear ion traps [3–6] and a three-dimensional quadrupole (Paul) ion trap (QIT) [7–12]. For the former, a 22-pole ion trap is the most popular [3–5], but an octapole ion trap was reported to be useful for this purpose [6]. Rizzo and co-workers are using the 22-pole ion trap, and have estimated the effective temperature of the ions to be 10 K [1,3]. These multi-pole linear ion traps provide ⇑ Corresponding authors. E-mail addresses: [email protected] (S.-i. Ishiuchi), [email protected]. ac.jp (M. Fujii).
a flat potential field around the center axis of the ion traps, which is thought to be important for a high cooling efficiency [13–15]. However, since the ions are distributed linearly along the center axis, it is not easy to interface the linear ion traps to a time-offlight mass spectrometer (TOF-MS). Another problem of a linear ion trap is that UV-UV hole burning (HB) spectroscopy, which gives conformer-selected UV spectra, is difficult to be applied for linear ion trap experiments. Conformation-selected spectroscopy is indispensable to investigate the structure and dynamics of large flexible molecules, such as peptides. Typical spectroscopic techniques used to distinguish conformers are IR-UV or UV-UV HB spectroscopies, in which conformers are discriminated by the differences in the vibrational or electronic transition energy, respectively [16–18]. However, the vibrational frequencies are generally not as sensitive as the electronic ones with respect to tiny conformational differences, such as rotamer [17–19]. For example, a neutral synephrine molecule (adrenaline analogue) has 6 conformers that consist of 3 pairs of cis- and trans-rotamers for OH orientations. Such cis- and trans-rotamers give almost the same IR spectra (the differences in the OH stretching vibrations are within 1 cm1) [19]. Therefore, it is difficult to conclude whether these are different species or not only from the IR-UV HB spectra. On the other hand, the vibrational-electronic transitions of the rotamer pairs are well separated by more than 10 cm1, so that the UV-UV hole burning spectra clearly separate all of the conformers spectroscopically without doubt [19,20]. Thus, the availability of UV-UV HB
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spectroscopy has a significant advantage in a structural study of large flexible molecules. To measure the UV-UV HB spectra, two tunable UV lasers are employed. The first UV laser (probe laser) is fixed at an electronic transition of a certain conformer, and fragment ions produced by predissociation in the electronically excited state are monitored. This signal intensity is proportional to the population of the conformer labeled by the probe laser. Then, the second UV laser (burn laser) is introduced before firing the probe laser, and is scanned. When the wavelength of the burn laser corresponds to the electronic transitions of the labeled conformer, the population of the labeled conformer decreases, and the signal intensity of the fragment ion due to the probe laser decreases. Thus, the electronic spectrum of the labeled conformer by the probe laser can be measured as decreases of the fragment ion intensity, which is the UV-UV HB spectrum. A problem of this method is the fact that the burn laser also produces fragment ions, and thus the fragment ions due to the burn laser have to be separated from those due to the probe laser. Linear ion traps can trap a mass-selected ion by adding a DC offset to the RF voltage, i.e., such as a quadrupole mass spectrometer (Q-MS), in principle. If one can easily use the mass-select mode with the linear ion traps, the discrimination of fragment ions due to the burn and probe lasers can be achieved by switching the mode from mass-select (only the parent ion can be trapped) to not mass-select (also fragment ions can be trapped) between the irradiation of the burn and probe lasers. However, since it is difficult to generalize the stable region for the higher order multi poles [15], the mass-select mode is difficult to be performed. Therefore, UV-UV HB spectroscopy is hardly achieved by the linear ion traps. The QIT has advantages for the combinations to TOF-MS and UV-UV HB spectroscopy. The QIT traps ions within a small spatial region; it is then feasible to interface to TOF-MS [21]. Another advantage of QIT is that UV-UV HB spectroscopy can be applied to trapped ions by using several techniques [22–25]. One of those is the ‘‘tickle” RF technique [24,26]. By applying a ‘‘tickle” RF signal to the end cap electrode of the QIT, a mass-specified ion, which is determined by the frequency of the ‘‘tickle” RF, can be pushed out from the QIT. By applying the ‘‘tickle” RF between the burn and probe lasers, the fragment ions produced by the burn laser can be removed from the QIT, and only fragments produced by the probe laser can be detected. However, the QIT has a problem concerning the cooling efficiency. It is thought that the cooling efficiency of the cryogenic QIT is less than the multi-pole linear ion traps, because the QIT does not have a field-free region, except for the center point. This structure of the field heats up trapped ions by excessive collisions with the buffer gas (RF heating). Indeed, even though the QIT, itself, is cooled down at 10 K, it has been reported that the typical temperature of trapped ions in QIT is 50 K, which is not sufficient to reduce hot bands [10]. RF heating appears to be an established interpretation, but we also think about another factor that also induces higher temperature in QIT. It is the temperature of the buffer gas. The buffer gas is cooled down by thermal contact with the ultra-cold ion trap and its surroundings. For linear ion traps, there is large space between the electrodes, and the ion trap is usually placed in a copper box whose temperature is the same as that of the ion trap. In this structure, the buffer gas can be cooled down mainly by thermal contact with the copper box rather than the electrodes. On the other hand, since the shape of the QIT is closed, the buffer gas is cooled down by the electrodes of the QIT, themselves. Then, the cooling efficiency of the buffer gas in the QIT could be strongly affected by the thermal conductivity of the material of the electrodes. Generally, the electrodes of the QIT are made from stainless-steel. However, the thermal conductivity of stainlesssteel (SUS304) is only 0.36 Wm1 K1 at 5 K [27], which is as low
as that of wood, and thus cannot be expected to cool down the buffer gas sufficiently. Recently, it was suggested that the cooling efficiency might be improved by avoiding heat flow to the QIT, for example, by surrounding the QIT with a copper box similar to linear ion traps [11]. This implies that the buffer gas is cooled down by the copper parts because the thermal conductivity of copper is 3.9 102 Wm1 K1 at 5 K [27], which is significantly higher than that of stainless-steel. If so, by making the QIT electrodes of copper, the cooling efficiency should be drastically improved. In this work, we built a QIT by using copper electrodes, and found a drastic improvement of the cooling efficiency, which has been spectroscopically proven by the UV photo-dissociation spectrum of protonated tyrosine (TyrH+). This copper QIT allows us to apply UV-UV HB spectroscopy to cold ions. Here, we have measured the UV-UV HB spectra of the TyrH+ for the first time, and established the number of conformers spectroscopically. The UV photo-dissociation spectrum and IR spectra obtained by IR-UV double resonance spectroscopy of cold TyrH+ have been reported by Rizzo and co-workers by using a 22-pole cold ion trap [28]. They revealed the co-existence of four conformers (two pairs of OH rotamers); however, they could not assign the absolute structures concerning to the OH orientations because of slight difference of vibrational frequencies. This shows the limitation of the vibrational spectroscopy for such rotamers. In this work, we discussed the structural assignments of TyrH+, including the OH orientation based on the conformer-selected UV spectra, and calculated the Franck-Condon spectra. 2. Experimental Fig. 1 shows a schematic diagram of our ESI/cold QIT tandem mass spectrometer. The ESI source was purchased from Bruker. A glass capillary was heated up at 60 °C by hot dry air. Through the capillary, ions were introduced to a vacuum (1 Torr). Through a skimmer, the ions were introduced to a higher vacuum region, and propagated to a Q-MS (Extrel) by a blade-type hexapole ion guide, which was driven by a 4 MHz RF generator (CGC Instruments). The mass-selected ions were introduced to a quadrupole ion deflector, which bent the ion trajectory by a right angle, and removed any neutral species. The ions were guided by an octapole ion guide driven by a 1.6 MHz RF generator (CGC Instruments) to a bare QIT (without a copper box) mounted on the head of a closedcycle refrigerator (Sumitomo: RDK-408D2). Between the octapole ion guide and the QIT, an ion lens was placed, which gated the ion beam at 20 Hz repetition by a pulsed voltage. Helium buffer gas (at room temperature) was introduced to the QIT through a pulsed nozzle (Parker: General Valve Series 9 driven by IOTA-I). The electrical pulse duration to open the pulsed nozzle was 120 ls. When opening the pulsed nozzle at time 0, the ion beam was injected to the QIT for 35 ms. One may expect that the duration of ion-injection affects the cooling efficiency of the trapped ion, because He buffer gas is pulse-injected at only the beginning of the ion injection, and its cooling effect may not continue until the end of the ion injection. We varied the duration of ion-injection from 10 to 35 ms; however, no effect on the cooling efficiency was found. At 38 ms, pulse of a tunable UV laser (frequency-doubled dye laser pumped by a nanosecond Nd3+: YAG laser operated at 20 Hz) was introduced to the QIT, and induced photo dissociation of the trapped ions. At 40 ms, the RF voltage of the QIT was turned off, and the ions were ejected to TOF-MS by a DC pulse (650 V) applied to the exit end-cap electrode of the QIT. The TOF-MS was floated at 4 kV. The ions were detected by a dynode converter detector. The signal was amplified tenfold by a preamplifier (NF: BX-31A) and recorded by a fast digitizer board (National Instruments: PXIe-5160) as a
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ion deflector octapole ion guide
QIT
Q-MS
burn laser TOF-MS
probe laser
hexapole ion guide
drift tube
pulsed valve
emitter
dynode converter
He injection
skimmer glass capillary
ion injection burn fire tickle RF probe fire ion eject 0
5
10
30
35
40
45
50
time / ms Fig. 1. Schematic diagram of the ESI/cold QIT tandem mass spectrometer and a timing chart of a one cycle measurement.
function of the wavelength of the UV laser. For UV-UV HB spectroscopy, the burn laser was introduced at 36 ms and a ‘‘tickle” RF pulse generated by a function generator (NF: WF1974), which removed the fragment ion produced by the burn laser, was applied to the entrance end cap of the QIT for 1 ms duration from 36 ms. The burn laser was operated at 10 Hz repetition, and thus the probe-only signal and the HB signal were obtained alternatively. By dividing the latter by the former, the depletion ratio was obtained. Fig. 2 shows a photograph of the QIT employed in this work. Three electrodes, the entrance and exit end caps and the ring electrode were made of oxygen-free copper (C1020), and were coated
by gold with a 4 lm thickness. Each electrode was insulated by sapphire cylinders. Both end caps had 3 mm holes in order to introduce and extract ions. The ring electrode had holes of 2 mm diameter on the lateral and bottom sides so as to introduce laser beams and the buffer gas. This QIT was produced by ADCAP VACUUM TECHNOLOGY Co., Ltd. The RF generator used to operate the QIT was purchased from Jordan TOF Products, Inc. (D-1203). Tyrosine was purchased from Aldrich and was used without any further purifications. The tyrosine was dissolved at a concentration of 105 M to methanol including 0.5% formic acid. The solution was supplied to the ESI emitter at 5 lL min1 by a syringe pump (ITO CORPORATION/Harvard Apparatus). 3. Computational To estimate the rotational temperature, the rotational envelope of the 0–0 band of conformer B [10] was simulated by using the PGOPHER program [29]. To obtain the rotational constants in the S0 state, geometrical optimization was carried out at the RIB3LYP-D3/def2-TZVP level by using the TURBOMOLE program (ver. 7.0) [30]. The rotational constants of the S1 state and the transition moment of the S1-S0 transition were calculated at the same level; however, they were optimized by fitting the rotational contour with PGOPHER. Each rotational line was convoluted by a Lorentzian, whose full width at half maximum was set to 0.3 cm1. Also, for simulating the Franck-Condon (FC) spectra, the PGOPHER program was employed. For FC simulations, geometrical optimization and vibrational analysis in the S0 and S1 states were carried out at the RI-CC2/aug-cc-pVDZ level by using the TURBOMOLE program. 4. Results and discussion 4.1. Effective temperature of TyrH+
Fig. 2. Photograph of the copper QIT.
At first, we measured the UV photo-dissociation spectrum by using a stainless-steel QIT purchased from Jordan TOF Products, Inc. (C-1251). Fig. 3a shows the UV photo-dissociation spectrum of TyrH+ obtained by monitoring a fragment mass peak at 108 u,
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S.-i. Ishiuchi et al. / Journal of Molecular Spectroscopy xxx (2016) xxx–xxx
Fragment intensity / arb. units
4
a)
0
b)
0 35050
35100
35150 35200 Wavenumber / cm-1
35250
35300
Fig. 3. UV photo-dissociation spectrum of protonated tyrosine trapped in (a) a stainless-steel QIT and (b) a copper QIT.
which corresponds to a fragment of Ca-Cb cleavage [31,32]. By comparing it with the UV spectrum reported by Rizzo and coworkers, who used a 22-pole ion trap, it was found that additional small peaks were observed in our spectrum, and that the bandwidth of our spectrum was wider than the reported one. Thus, the small bands observed in our spectrum were assigned to hot bands. Our spectrum is quite similar to that reported by Kim and co-workers, who used a commercial stainless-steel QIT. They estimated the rotational temperature by simulating the rotational contour of the 0–0 band of conformer B observed at 35,111 cm1. We also estimated the rotational temperature by simulating the rotational contour of the 0–0 band of conformer B. By the fitting, the rotational temperature was estimated to be 45.7 K (Fig. 4a). This temperature is similar to that reported by Kim and co-workers. On the other hand, to estimate the vibrational temperature, Kim and co-workers measured the intensity ratio of a hot band of conformer B with respect to its 0–0 transition intensity. That ratio varies by the FC factor; thus, they calculated it to determine the population ratio. Rizzo and co-workers also estimated the vibrational temperature of TyrH+; they measured the intensity ratio of a hot band (A0 1) and a vibronic band (A1 0) of conformer A (see Fig. 5). Since the FC factor should be similar between them,
Fragment intensity / arb. units
a) Trot = 45.7 K 0
b) Trot = 14.1 K 0 35105
35110 35115 Wavenumber / cm-1
35120
Fig. 4. Rotational contour of the 0–0 transition of conformer B observed in the UV photo-dissociation spectrum by using (a) a stainless-steel QIT and (b) a copper QIT. Rotational constants (cm1) in S0 and S1 states were fixed to A00 = 0.0794, B00 = 0.0115, C00 = 0.0109 and A0 = 0.0765, B0 = 0.0109, C0 = 0.0109, respectively. The transition moment was hS1|a|S0i = 0.0367, hS1|b|S0i = 0.175, hS1|c|S0i = 0.0266. Each rotational line was convoluted by a Lorentzian, whose full width of half maximum was 0.3 cm1.
the FC factors are cancelled out from the intensity ratio. Thus, one does not need to know the FC factor in this method. We estimated the vibrational temperature according to the same method, and obtained 51.1 K (Fig. 5a). This value is close to the rotational temperature, which suggests that the trapped ion is almost in a thermal equilibrium state, and both the vibrational and rotational temperatures can be regarded as being the temperature of the ions. Therefore, the temperature of the TyrH+ trapped in the stainlesssteel QIT is estimated to be 45–50 K. Incidentally, the temperature of the QIT, itself, was 4.6 K. We then changed the stainless-steel QIT to a copper QIT, and measured the UV spectrum of TyrH+ (Fig. 3b). The spectrum drastically changed; the hot bands were significantly weakened, and the bandwidth of each band became narrower. These results clearly demonstrate that the cooling efficiency was drastically improved. By fitting the rotational contour of the 0–0 band of conformer B, and measuring the band intensity ratio of A0 1 and A1 0, the rotational and vibrational temperatures were estimated to be 14.1 and 13.4 K, respectively (Figs. 4b and 5b). Thus, the temperature of the TyrH+ trapped in the copper QIT is 13 K. The temperature of the QIT, itself, was the same as that of the stainless-steel QIT. This also supports the importance of the thermal conductivity for buffer gas cooling. The ion temperature by using the copper QIT is still 3 K higher than that by using multi-pole linear ion traps (10.3 K in 22-pole [1], 9.1 K in octapole [6]). This may be due to the RF heating effect. However, ions at 13 K have only a 10% population, even at a lowfrequency vibrational level of 20 cm1, which corresponds to the typical lowest vibrational frequency of proteins [33]. Thus this cooling efficiency may be sufficient to study cold large molecular ions with the conformer-selected laser spectroscopy. 4.2. UV-UV HB spectroscopy of protonate tyrosine To measure the UV-UV HB spectra, we must separate the photo fragments induced by the burn laser. We therefore applied a RF pulse to push them out from the QIT before irradiating the remaining ion with the probe laser (‘‘tickle” RF method [24,26]). Since we monitored a fragment at m/z = 108, which was generated by the probe laser, in order to remove the same fragment generated by the burn laser, the frequency of the ‘‘tickle” RF pulse was tuned to 109.236 kHz (amplitude was 3 V). By applying this ‘‘tickle” RF pulse for 1 ms, the fragment ion at m/z = 108 generated by the burn laser could be completely removed from the QIT. Fig. 6 shows the UV-UV HB spectra and the UV photodissociation spectrum (bottom) of TyrH+. By probing bands observed at 35081.7, 35111.3, 35186.0 and 35234.1 cm1, four HB spectra AD were measured, respectively. Since these bands are the red-most band in each HB spectrum, they can be clearly assigned to the 0–0 transition of each conformer. All bands observed in the UV photo-dissociation spectrum were observed in either of the AD HB spectra, and it is thus concluded that the UV photo-dissociation spectrum contains four conformers. To compare the spectral pattern, the horizontal axes were arranged by the 0–0 transition (Fig. 7). According to the spectral similarity, the UV-UV HB spectra can be classified into two groups, (A, C) and (B, D). Generally, the vibronic band patterns are roughly similar between phenolic OH rotamers [18,19]. Thus, it is expected that the two conformers of each group are in a relationship of the phenolic OH rotamers. This expectation coincides to the structural assignments by the IR spectra of the X-H stretching region [28]. The simulated FC spectra of the four conformers of TyrH+ by using the PGOPHER program have already been reported by Féraud et al. [34] By comparing them with the observed UV-UV HB spectra, it was found that the simulated spectrum of the conformer B (rot/syn according to their notation) does not show a good
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B0←0
a)
Fragment intensity / arb. units
A0←0
A1←0
Tvib = 51.1 K A0←1 0
B0←0
b) Tvib = 13.4 K
A0←0
A1←0
A0←1 ×20 0 35020
35040
35060
35080 35100 Wavenumber / cm-1
35120
35140
Fig. 5. Lower energy region of the UV photo-dissociation spectrum of protonated tyrosine trapped in (a) a stainless-steel QIT and (b) a copper QIT. The intensities of each vibronic band were estimated by fitting by a Gaussian (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(νP + νB) signal / νP signal
1 0 1
D C
0 1
B 0 1
A
Fragment intensity / arb. units
0
0 35050
35100
35150
35200
35250
35300
Wavenumber / cm-1 Fig. 6. UV-UB HB spectra of four conformers (AD) and the UV photo-dissociation spectrum of protonated tyrosine. Each UV-UV HB spectrum was measured by fixing the probe laser to each band represented by dotted lines.
agreement with the observed one, while others comparatively well reproduce the observed ones. To simulate the FC spectra by using the PGOPHER program, the maximum vibrational quantum numbers of each mode in the excited state should be set manually. There is thus a possibility of missing for that setting in the simulation of conformer B. We thus also calculated the FC spectra by using the PGOPHER program. Geometrical optimization and vibrational analysis prior to the FC simulation were carried out at the same level as that of Féraud et al. Fig. 7 also shows the newly calculated FC spectra of the four conformers. The frequencies of the lowest 10 modes are listed in
Table 1. By comparing the FC spectrum of conformer B of this work with the reported one [34], it was found that the latter does not include modes 3 and 5. In conformers A and C, the 21 transition appears, while not it but the 31 transition appears in conformers B and D, which can explain the difference between the two groups, (A, C) and (B, D). In the FC spectra of A and C, the intensity pattern of 11 and 21 is different, i.e., the former is stronger than the latter in A, while they are comparative in C. Since the UV-UV HB spectra are saturated, the intensity patterns are also calibrated from the UV photo-dissociation spectrum. From these spectra, it is found that the 11 transition of conformer A is stronger than 21. Thus,
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A 00 11 21
3
1251, 2171 1151 4151 13 1231 1 1 1 7 5 1 71 1 1 81 1131 1 4 1241 61 1331 1161
1121 2 41 1 1
C 00 11 2
1
1 1
12 12 22 41
1151 1 1 1 1 1 2 6 1 112141 7 1 6 1 1 8 1 1 1171 2 7 2 5 1 6
1141 1122 2141 51 1221
B 00
61 11
31
41 12
1141 314
1 1
13
1151 1 1 71 1 6
1 1 5
4151
1171
4161
D 00 1
11
0
50
3
1
51 6
41
81 416 1 4171
11511161 1171 4151
1141
100
71
150
200
250
Wavenumber / cm-1 Fig. 7. UV-UV HB spectra of the four conformers aligned by 0–0 transition energy and calculated FC spectra of each conformer.
Table 1 Vibrational frequencies (cm1) of the lowest 10 modes of each conformer in the S1 (pp⁄) state calculated at RI-CC2/aug-cc-pVDZ level. Mode
Conf. A
Conf. B
Conf. C
Conf. D
1 2 3 4 5 6 7 8 9 10
44.2 47.3 80.3 97.9 167.2 198.1 208.1 279.9 286.8 322.5
44.7 52.2 64.2 88.2 165.4 171.1 200.8 282.3 286.1 317.8
43.6 47.4 81.0 102.0 161.3 187.5 206.4 277.1 285.7 318.8
41.1 51.1 65.7 87.7 160.5 169.1 189.4 279.0 285.9 308.4
the structures of conformers A and C are determined as shown in Fig. 7. In the observed spectra of B and D, a difference of the 11 transition energy is found between them. That of B is 00 + 44.1 cm1, while it is 40.3 cm1 for D. This tendency is well reproduced by the calculation (see Table 1). Thus, the absolute OH orientation in the (B, D) pair is concluded to be shown in Fig. 7. 5. Conclusion To decrease the temperature of ions trapped in the QIT, we made it from copper. When a normal QIT made from stainlesssteel was employed, the temperature of TyrH+ was estimated to be 45–50 K, based on the rotational contour and the hot band intensity observed in the UV photo-dissociation spectrum of TyrH+. By replacing it by a copper QIT, the intensity of the hot bands observed in the UV spectrum drastically decreased. The temperature of the TyrH+ trapped in the copper QIT was estimated to be
13 K. So far, the reason for the low cooling efficiency by the cryogenic QIT has been thought to be due to RF heating, because it does not have a free field region, except for the center point. However, the result of this work clearly shows that the main reason for it is the material of the QIT. Interestingly, the temperature of the copper QIT, itself, was the same as that of the stainless-steel QIT. Nonetheless, the temperature of ions trapped in the former was much lower than that of the latter. This result indicates that the temperature of the buffer gas is different between them, which can be explained by the difference in the thermal conductivity, i.e., the thermal conductivity of stainless-steel is much lower than that of copper at the ultra-cold temperature, and that the low thermal conductivity causes a low cooling efficiency of the buffer gas. The cooling efficiency of cryogenic QIT can be improved significantly by making it from copper, which enables us to obtain conformer-selected UV spectra of cold ions by using UV-UV HB spectroscopy under a sufficiently cold condition. Its advantage was demonstrated by the UV-UV HB spectra of TyrH+. Four different UV-UV HB spectra were measured and the co-existence of four conformers was confirmed spectroscopically. From the spectral similarity, it was confirmed that conformers A and C, and conformers B and D are the phenolic OH rotamer pair, respectively. By comparing with the calculated FC spectra, the absolute phenolic OH orientations have been determined. Acknowledgements We are grateful for Prof. Gilles Grégoire, Prof. Yoshiya Inokuchi and Prof. Nam Joon Kim for giving us fruitful advice and inspiration to improve the cooling efficiency of the QIT. Mr. Kunihiro Aoshima and Mr. Hiroyuki Miyamoto at ADCAP VACUUM TECHNOLOGY CO, LTD (http://www.adcap-vacuum.com) provided help to us to make
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the copper QIT. We deeply thank them. This work was supported in part by a Grant-in-Aid for Scientific Research KAKENHI on Innovative Area (2503): ‘‘Studying the Function of Soft Molecular Systems by the Concerted Use of Theory and Experiment’’, KAKENHI in the priority area ‘‘Molecular Science for Supra Functional Systems’’, a Grant-in-Aid for Young Scientists KAKENHI, and the Cooperative Research Program of ‘‘Network Joint Research Center for Materials and Devices’’, from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan.
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[14] D. Gerlich, The study of cold collisions using ion guides and traps, in: I.W.M. Smith (Ed.), Low Temperatures and Cold Molecules, World Scientific Publishing, Singapore, 2008, pp. 121–174. [15] D. Gerlich, Inhomogeneous electrical radio frequency fields: a versatile tool for the study of processes with slow ions, in: C.Y. Ng, M. Baer, I. Prigogine, S.A. Rice (Eds.), Advances in Chemical Physics: State-Selected and State-To-State Ion-Molecule Reaction Dynamics, Part 1. Experiment, John Wiley & Sons, 1992. [16] E.C. Stanca-Kaposta, P. Carcabal, E.J. Cocinero, P. Hurtado, J.P. Simons, J. Phys. Chem. B 117 (2013) 8135. [17] W.Y. Sohn, S. Ishiuchi, M. Miyazaki, J. Kang, S. Lee, A. Min, M.Y. Choi, H. Kang, M. Fujii, Phys. Chem. Chem. Phys. 15 (2013) 957. [18] Y. Shimozono, K. Yamada, S. Ishiuchi, K. Tsukiyama, M. Fujii, Phys. Chem. Chem. Phys. 15 (2013) 5163. [19] S. Ishiuchi, T. Asakawa, H. Mitsuda, M. Miyazaki, S. Chakraborty, M. Fujii, J. Phys. Chem. A 115 (2011) 10363. [20] S. Ishiuchi, H. Sone, M. Fujii, Chem. Lett. 42 (2013) 1166. [21] S.M. Michael, M. Chien, D.M. Lubman, Rev. Sci. Instrum. 63 (1992) 4277. [22] C.M. Choi, D.H. Choi, J. Heo, N.J. Kim, S.K. Kim, Angew. Chem. Int. Ed. 51 (2012) 7297. [23] G. Feraud, C. Dedonder, C. Jouvet, Y. Inokuchi, T. Haino, R. Sekiya, T. Ebata, J. Phys. Chem. Lett. 5 (2014) 1236. [24] H. Kang, G. Féraud, C. Dedonder-Lardeux, C. Jouvet, J. Phys. Chem. Lett. 5 (2014) 2760. [25] M. Broquier, S. Soorkia, G. Gregoire, Phys. Chem. Chem. Phys. 17 (2015) 25854. [26] R.E. March, J. Mass Spectrom. 32 (1997) 351. [27] NIST Cryogenic Materials database, 2010. [28] J.A. Stearns, S. Mercier, C. Seaiby, M. Guidi, O.V. Boyarkin, T.R. Rizzo, J. Am. Chem. Soc. 129 (2007) 11814. [29] C.M. Western, J. Quant. Spectrosc. Radiat. Transf. (2016), http://dx.doi.org/ 10.1016/j.jqsrt.2016.04.010 (in press). [30] R. Ahlrichs, M. Bär, M. Häser, H. Horn, C. Kölmel, Chem. Phys. Lett. 162 (1989) 165. [31] H. Kang, C. Jouvet, C. Dedonder-Lardeux, S. Martrenchard, G. Gregoire, C. Desfrancois, J.P. Schermann, M. Barat, J.A. Fayeton, Phys. Chem. Chem. Phys. 7 (2005) 394. [32] A. Svendsen, U.J. Lorenz, O.V. Boyarkin, T.R. Rizzo, Rev. Sci. Instrum. 81 (2010) 073107. [33] P.C. Painter, L.E. Mosher, C. Rhoads, Biopolymers 21 (1982) 1469. [34] G. Feraud, M. Broquier, C. Dedonder, C. Jouvet, G. Gregoire, S. Soorkia, J. Phys. Chem. A 119 (2015) 5914.
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High-cooling-efficiency cryogenic quadrupole ion trap and UV-UV hole burning spectroscopy of protonated tyrosine Shun-ichi Ishiuchi ⇑, Hiromichi Wako, Daichi Kato, Masaaki Fujii ⇑ Laboratory for Chemistry and Life Science, Institute of Innovation Research, Tokyo Institute of Technology, 4259, Nagatsuta-cho, Midori-ku, Yokohama, Japan
a r t i c l e
i n f o
Article history: Received 27 September 2016 In revised form 12 October 2016 Accepted 17 October 2016 Available online xxxx Keywords: Cold ion spectroscopy Electro spray ionization Cryogenic ion trap Gas phase spectroscopy of biomolecules Double resonance laser spectroscopy
a b s t r a c t The cooling efficiency of a cryogenic three-dimensional quadrupole ion trap (QIT) is drastically improved by using copper electrodes instead of conventional stainless-steel ones. The temperature of trapped ions (protonated tyrosine TyrH+) was estimated based on the ultraviolet (UV) photo-dissociation spectra. The UV spectrum of TryH+ shows almost no hot bands, and thus the high cooling efficiency of the copper ion trap was proven. The temperature was also estimated by simulating the observed band contour in the UV spectra, which is determined by the population in the rotationally excited levels. From the simulations, the temperature of TryH+ was estimated to be 13 K, while that in the stainless-steel QIT was 45–50 K. In addition, to demonstrate the advantage of the copper QIT, UV-UV hole burning (HB) spectra, i.e. conformation-selected UV spectra, were measured. It was confirmed that four different conformers, AD, coexist in the ultra-cold protonated tyrosine. By comparing with the calculated Franck-Condon spectra, their structural assignments were discussed, including the orientation of the OH group. Ó 2016 Published by Elsevier Inc.
1. Introduction The combination of electrospray ionization (ESI) and a cryogenic ion trap enables us to apply high-resolution laser spectroscopy to non-volatile and large molecular ions, such as biomolecules, and supra molecular systems [1]. This methodology can provide precise conformational separation and structural determination, and their possibilities are further enhanced by combining with ion mobility spectrometry (IMS) [2]. The key technique of this methodology is buffer gas cooling in the cryogenic ion trap, which is crucial to reduce population of higher-energy conformers. The cooling process of the ions consists of two steps. At first, the injected buffer gas is cooled down by thermal contact with a cryogenic ion trap; then, trapped ions are cooled down by collisions with the cold buffer gas. Currently, two types of ion traps are used for this purpose, mainly: linear ion traps [3–6] and a three-dimensional quadrupole (Paul) ion trap (QIT) [7–12]. For the former, a 22-pole ion trap is the most popular [3–5], but an octapole ion trap was reported to be useful for this purpose [6]. Rizzo and co-workers are using the 22-pole ion trap, and have estimated the effective temperature of the ions to be 10 K [1,3]. These multi-pole linear ion traps provide ⇑ Corresponding authors. E-mail addresses: [email protected] (S.-i. Ishiuchi), [email protected]. ac.jp (M. Fujii).
a flat potential field around the center axis of the ion traps, which is thought to be important for a high cooling efficiency [13–15]. However, since the ions are distributed linearly along the center axis, it is not easy to interface the linear ion traps to a time-offlight mass spectrometer (TOF-MS). Another problem of a linear ion trap is that UV-UV hole burning (HB) spectroscopy, which gives conformer-selected UV spectra, is difficult to be applied for linear ion trap experiments. Conformation-selected spectroscopy is indispensable to investigate the structure and dynamics of large flexible molecules, such as peptides. Typical spectroscopic techniques used to distinguish conformers are IR-UV or UV-UV HB spectroscopies, in which conformers are discriminated by the differences in the vibrational or electronic transition energy, respectively [16–18]. However, the vibrational frequencies are generally not as sensitive as the electronic ones with respect to tiny conformational differences, such as rotamer [17–19]. For example, a neutral synephrine molecule (adrenaline analogue) has 6 conformers that consist of 3 pairs of cis- and trans-rotamers for OH orientations. Such cis- and trans-rotamers give almost the same IR spectra (the differences in the OH stretching vibrations are within 1 cm1) [19]. Therefore, it is difficult to conclude whether these are different species or not only from the IR-UV HB spectra. On the other hand, the vibrational-electronic transitions of the rotamer pairs are well separated by more than 10 cm1, so that the UV-UV hole burning spectra clearly separate all of the conformers spectroscopically without doubt [19,20]. Thus, the availability of UV-UV HB
http://dx.doi.org/10.1016/j.jms.2016.10.011 0022-2852/Ó 2016 Published by Elsevier Inc.
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spectroscopy has a significant advantage in a structural study of large flexible molecules. To measure the UV-UV HB spectra, two tunable UV lasers are employed. The first UV laser (probe laser) is fixed at an electronic transition of a certain conformer, and fragment ions produced by predissociation in the electronically excited state are monitored. This signal intensity is proportional to the population of the conformer labeled by the probe laser. Then, the second UV laser (burn laser) is introduced before firing the probe laser, and is scanned. When the wavelength of the burn laser corresponds to the electronic transitions of the labeled conformer, the population of the labeled conformer decreases, and the signal intensity of the fragment ion due to the probe laser decreases. Thus, the electronic spectrum of the labeled conformer by the probe laser can be measured as decreases of the fragment ion intensity, which is the UV-UV HB spectrum. A problem of this method is the fact that the burn laser also produces fragment ions, and thus the fragment ions due to the burn laser have to be separated from those due to the probe laser. Linear ion traps can trap a mass-selected ion by adding a DC offset to the RF voltage, i.e., such as a quadrupole mass spectrometer (Q-MS), in principle. If one can easily use the mass-select mode with the linear ion traps, the discrimination of fragment ions due to the burn and probe lasers can be achieved by switching the mode from mass-select (only the parent ion can be trapped) to not mass-select (also fragment ions can be trapped) between the irradiation of the burn and probe lasers. However, since it is difficult to generalize the stable region for the higher order multi poles [15], the mass-select mode is difficult to be performed. Therefore, UV-UV HB spectroscopy is hardly achieved by the linear ion traps. The QIT has advantages for the combinations to TOF-MS and UV-UV HB spectroscopy. The QIT traps ions within a small spatial region; it is then feasible to interface to TOF-MS [21]. Another advantage of QIT is that UV-UV HB spectroscopy can be applied to trapped ions by using several techniques [22–25]. One of those is the ‘‘tickle” RF technique [24,26]. By applying a ‘‘tickle” RF signal to the end cap electrode of the QIT, a mass-specified ion, which is determined by the frequency of the ‘‘tickle” RF, can be pushed out from the QIT. By applying the ‘‘tickle” RF between the burn and probe lasers, the fragment ions produced by the burn laser can be removed from the QIT, and only fragments produced by the probe laser can be detected. However, the QIT has a problem concerning the cooling efficiency. It is thought that the cooling efficiency of the cryogenic QIT is less than the multi-pole linear ion traps, because the QIT does not have a field-free region, except for the center point. This structure of the field heats up trapped ions by excessive collisions with the buffer gas (RF heating). Indeed, even though the QIT, itself, is cooled down at 10 K, it has been reported that the typical temperature of trapped ions in QIT is 50 K, which is not sufficient to reduce hot bands [10]. RF heating appears to be an established interpretation, but we also think about another factor that also induces higher temperature in QIT. It is the temperature of the buffer gas. The buffer gas is cooled down by thermal contact with the ultra-cold ion trap and its surroundings. For linear ion traps, there is large space between the electrodes, and the ion trap is usually placed in a copper box whose temperature is the same as that of the ion trap. In this structure, the buffer gas can be cooled down mainly by thermal contact with the copper box rather than the electrodes. On the other hand, since the shape of the QIT is closed, the buffer gas is cooled down by the electrodes of the QIT, themselves. Then, the cooling efficiency of the buffer gas in the QIT could be strongly affected by the thermal conductivity of the material of the electrodes. Generally, the electrodes of the QIT are made from stainless-steel. However, the thermal conductivity of stainlesssteel (SUS304) is only 0.36 Wm1 K1 at 5 K [27], which is as low
as that of wood, and thus cannot be expected to cool down the buffer gas sufficiently. Recently, it was suggested that the cooling efficiency might be improved by avoiding heat flow to the QIT, for example, by surrounding the QIT with a copper box similar to linear ion traps [11]. This implies that the buffer gas is cooled down by the copper parts because the thermal conductivity of copper is 3.9 102 Wm1 K1 at 5 K [27], which is significantly higher than that of stainless-steel. If so, by making the QIT electrodes of copper, the cooling efficiency should be drastically improved. In this work, we built a QIT by using copper electrodes, and found a drastic improvement of the cooling efficiency, which has been spectroscopically proven by the UV photo-dissociation spectrum of protonated tyrosine (TyrH+). This copper QIT allows us to apply UV-UV HB spectroscopy to cold ions. Here, we have measured the UV-UV HB spectra of the TyrH+ for the first time, and established the number of conformers spectroscopically. The UV photo-dissociation spectrum and IR spectra obtained by IR-UV double resonance spectroscopy of cold TyrH+ have been reported by Rizzo and co-workers by using a 22-pole cold ion trap [28]. They revealed the co-existence of four conformers (two pairs of OH rotamers); however, they could not assign the absolute structures concerning to the OH orientations because of slight difference of vibrational frequencies. This shows the limitation of the vibrational spectroscopy for such rotamers. In this work, we discussed the structural assignments of TyrH+, including the OH orientation based on the conformer-selected UV spectra, and calculated the Franck-Condon spectra. 2. Experimental Fig. 1 shows a schematic diagram of our ESI/cold QIT tandem mass spectrometer. The ESI source was purchased from Bruker. A glass capillary was heated up at 60 °C by hot dry air. Through the capillary, ions were introduced to a vacuum (1 Torr). Through a skimmer, the ions were introduced to a higher vacuum region, and propagated to a Q-MS (Extrel) by a blade-type hexapole ion guide, which was driven by a 4 MHz RF generator (CGC Instruments). The mass-selected ions were introduced to a quadrupole ion deflector, which bent the ion trajectory by a right angle, and removed any neutral species. The ions were guided by an octapole ion guide driven by a 1.6 MHz RF generator (CGC Instruments) to a bare QIT (without a copper box) mounted on the head of a closedcycle refrigerator (Sumitomo: RDK-408D2). Between the octapole ion guide and the QIT, an ion lens was placed, which gated the ion beam at 20 Hz repetition by a pulsed voltage. Helium buffer gas (at room temperature) was introduced to the QIT through a pulsed nozzle (Parker: General Valve Series 9 driven by IOTA-I). The electrical pulse duration to open the pulsed nozzle was 120 ls. When opening the pulsed nozzle at time 0, the ion beam was injected to the QIT for 35 ms. One may expect that the duration of ion-injection affects the cooling efficiency of the trapped ion, because He buffer gas is pulse-injected at only the beginning of the ion injection, and its cooling effect may not continue until the end of the ion injection. We varied the duration of ion-injection from 10 to 35 ms; however, no effect on the cooling efficiency was found. At 38 ms, pulse of a tunable UV laser (frequency-doubled dye laser pumped by a nanosecond Nd3+: YAG laser operated at 20 Hz) was introduced to the QIT, and induced photo dissociation of the trapped ions. At 40 ms, the RF voltage of the QIT was turned off, and the ions were ejected to TOF-MS by a DC pulse (650 V) applied to the exit end-cap electrode of the QIT. The TOF-MS was floated at 4 kV. The ions were detected by a dynode converter detector. The signal was amplified tenfold by a preamplifier (NF: BX-31A) and recorded by a fast digitizer board (National Instruments: PXIe-5160) as a
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ion deflector octapole ion guide
QIT
Q-MS
burn laser TOF-MS
probe laser
hexapole ion guide
drift tube
pulsed valve
emitter
dynode converter
He injection
skimmer glass capillary
ion injection burn fire tickle RF probe fire ion eject 0
5
10
30
35
40
45
50
time / ms Fig. 1. Schematic diagram of the ESI/cold QIT tandem mass spectrometer and a timing chart of a one cycle measurement.
function of the wavelength of the UV laser. For UV-UV HB spectroscopy, the burn laser was introduced at 36 ms and a ‘‘tickle” RF pulse generated by a function generator (NF: WF1974), which removed the fragment ion produced by the burn laser, was applied to the entrance end cap of the QIT for 1 ms duration from 36 ms. The burn laser was operated at 10 Hz repetition, and thus the probe-only signal and the HB signal were obtained alternatively. By dividing the latter by the former, the depletion ratio was obtained. Fig. 2 shows a photograph of the QIT employed in this work. Three electrodes, the entrance and exit end caps and the ring electrode were made of oxygen-free copper (C1020), and were coated
by gold with a 4 lm thickness. Each electrode was insulated by sapphire cylinders. Both end caps had 3 mm holes in order to introduce and extract ions. The ring electrode had holes of 2 mm diameter on the lateral and bottom sides so as to introduce laser beams and the buffer gas. This QIT was produced by ADCAP VACUUM TECHNOLOGY Co., Ltd. The RF generator used to operate the QIT was purchased from Jordan TOF Products, Inc. (D-1203). Tyrosine was purchased from Aldrich and was used without any further purifications. The tyrosine was dissolved at a concentration of 105 M to methanol including 0.5% formic acid. The solution was supplied to the ESI emitter at 5 lL min1 by a syringe pump (ITO CORPORATION/Harvard Apparatus). 3. Computational To estimate the rotational temperature, the rotational envelope of the 0–0 band of conformer B [10] was simulated by using the PGOPHER program [29]. To obtain the rotational constants in the S0 state, geometrical optimization was carried out at the RIB3LYP-D3/def2-TZVP level by using the TURBOMOLE program (ver. 7.0) [30]. The rotational constants of the S1 state and the transition moment of the S1-S0 transition were calculated at the same level; however, they were optimized by fitting the rotational contour with PGOPHER. Each rotational line was convoluted by a Lorentzian, whose full width at half maximum was set to 0.3 cm1. Also, for simulating the Franck-Condon (FC) spectra, the PGOPHER program was employed. For FC simulations, geometrical optimization and vibrational analysis in the S0 and S1 states were carried out at the RI-CC2/aug-cc-pVDZ level by using the TURBOMOLE program. 4. Results and discussion 4.1. Effective temperature of TyrH+
Fig. 2. Photograph of the copper QIT.
At first, we measured the UV photo-dissociation spectrum by using a stainless-steel QIT purchased from Jordan TOF Products, Inc. (C-1251). Fig. 3a shows the UV photo-dissociation spectrum of TyrH+ obtained by monitoring a fragment mass peak at 108 u,
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Fragment intensity / arb. units
4
a)
0
b)
0 35050
35100
35150 35200 Wavenumber / cm-1
35250
35300
Fig. 3. UV photo-dissociation spectrum of protonated tyrosine trapped in (a) a stainless-steel QIT and (b) a copper QIT.
which corresponds to a fragment of Ca-Cb cleavage [31,32]. By comparing it with the UV spectrum reported by Rizzo and coworkers, who used a 22-pole ion trap, it was found that additional small peaks were observed in our spectrum, and that the bandwidth of our spectrum was wider than the reported one. Thus, the small bands observed in our spectrum were assigned to hot bands. Our spectrum is quite similar to that reported by Kim and co-workers, who used a commercial stainless-steel QIT. They estimated the rotational temperature by simulating the rotational contour of the 0–0 band of conformer B observed at 35,111 cm1. We also estimated the rotational temperature by simulating the rotational contour of the 0–0 band of conformer B. By the fitting, the rotational temperature was estimated to be 45.7 K (Fig. 4a). This temperature is similar to that reported by Kim and co-workers. On the other hand, to estimate the vibrational temperature, Kim and co-workers measured the intensity ratio of a hot band of conformer B with respect to its 0–0 transition intensity. That ratio varies by the FC factor; thus, they calculated it to determine the population ratio. Rizzo and co-workers also estimated the vibrational temperature of TyrH+; they measured the intensity ratio of a hot band (A0 1) and a vibronic band (A1 0) of conformer A (see Fig. 5). Since the FC factor should be similar between them,
Fragment intensity / arb. units
a) Trot = 45.7 K 0
b) Trot = 14.1 K 0 35105
35110 35115 Wavenumber / cm-1
35120
Fig. 4. Rotational contour of the 0–0 transition of conformer B observed in the UV photo-dissociation spectrum by using (a) a stainless-steel QIT and (b) a copper QIT. Rotational constants (cm1) in S0 and S1 states were fixed to A00 = 0.0794, B00 = 0.0115, C00 = 0.0109 and A0 = 0.0765, B0 = 0.0109, C0 = 0.0109, respectively. The transition moment was hS1|a|S0i = 0.0367, hS1|b|S0i = 0.175, hS1|c|S0i = 0.0266. Each rotational line was convoluted by a Lorentzian, whose full width of half maximum was 0.3 cm1.
the FC factors are cancelled out from the intensity ratio. Thus, one does not need to know the FC factor in this method. We estimated the vibrational temperature according to the same method, and obtained 51.1 K (Fig. 5a). This value is close to the rotational temperature, which suggests that the trapped ion is almost in a thermal equilibrium state, and both the vibrational and rotational temperatures can be regarded as being the temperature of the ions. Therefore, the temperature of the TyrH+ trapped in the stainlesssteel QIT is estimated to be 45–50 K. Incidentally, the temperature of the QIT, itself, was 4.6 K. We then changed the stainless-steel QIT to a copper QIT, and measured the UV spectrum of TyrH+ (Fig. 3b). The spectrum drastically changed; the hot bands were significantly weakened, and the bandwidth of each band became narrower. These results clearly demonstrate that the cooling efficiency was drastically improved. By fitting the rotational contour of the 0–0 band of conformer B, and measuring the band intensity ratio of A0 1 and A1 0, the rotational and vibrational temperatures were estimated to be 14.1 and 13.4 K, respectively (Figs. 4b and 5b). Thus, the temperature of the TyrH+ trapped in the copper QIT is 13 K. The temperature of the QIT, itself, was the same as that of the stainless-steel QIT. This also supports the importance of the thermal conductivity for buffer gas cooling. The ion temperature by using the copper QIT is still 3 K higher than that by using multi-pole linear ion traps (10.3 K in 22-pole [1], 9.1 K in octapole [6]). This may be due to the RF heating effect. However, ions at 13 K have only a 10% population, even at a lowfrequency vibrational level of 20 cm1, which corresponds to the typical lowest vibrational frequency of proteins [33]. Thus this cooling efficiency may be sufficient to study cold large molecular ions with the conformer-selected laser spectroscopy. 4.2. UV-UV HB spectroscopy of protonate tyrosine To measure the UV-UV HB spectra, we must separate the photo fragments induced by the burn laser. We therefore applied a RF pulse to push them out from the QIT before irradiating the remaining ion with the probe laser (‘‘tickle” RF method [24,26]). Since we monitored a fragment at m/z = 108, which was generated by the probe laser, in order to remove the same fragment generated by the burn laser, the frequency of the ‘‘tickle” RF pulse was tuned to 109.236 kHz (amplitude was 3 V). By applying this ‘‘tickle” RF pulse for 1 ms, the fragment ion at m/z = 108 generated by the burn laser could be completely removed from the QIT. Fig. 6 shows the UV-UV HB spectra and the UV photodissociation spectrum (bottom) of TyrH+. By probing bands observed at 35081.7, 35111.3, 35186.0 and 35234.1 cm1, four HB spectra AD were measured, respectively. Since these bands are the red-most band in each HB spectrum, they can be clearly assigned to the 0–0 transition of each conformer. All bands observed in the UV photo-dissociation spectrum were observed in either of the AD HB spectra, and it is thus concluded that the UV photo-dissociation spectrum contains four conformers. To compare the spectral pattern, the horizontal axes were arranged by the 0–0 transition (Fig. 7). According to the spectral similarity, the UV-UV HB spectra can be classified into two groups, (A, C) and (B, D). Generally, the vibronic band patterns are roughly similar between phenolic OH rotamers [18,19]. Thus, it is expected that the two conformers of each group are in a relationship of the phenolic OH rotamers. This expectation coincides to the structural assignments by the IR spectra of the X-H stretching region [28]. The simulated FC spectra of the four conformers of TyrH+ by using the PGOPHER program have already been reported by Féraud et al. [34] By comparing them with the observed UV-UV HB spectra, it was found that the simulated spectrum of the conformer B (rot/syn according to their notation) does not show a good
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B0←0
a)
Fragment intensity / arb. units
A0←0
A1←0
Tvib = 51.1 K A0←1 0
B0←0
b) Tvib = 13.4 K
A0←0
A1←0
A0←1 ×20 0 35020
35040
35060
35080 35100 Wavenumber / cm-1
35120
35140
Fig. 5. Lower energy region of the UV photo-dissociation spectrum of protonated tyrosine trapped in (a) a stainless-steel QIT and (b) a copper QIT. The intensities of each vibronic band were estimated by fitting by a Gaussian (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(νP + νB) signal / νP signal
1 0 1
D C
0 1
B 0 1
A
Fragment intensity / arb. units
0
0 35050
35100
35150
35200
35250
35300
Wavenumber / cm-1 Fig. 6. UV-UB HB spectra of four conformers (AD) and the UV photo-dissociation spectrum of protonated tyrosine. Each UV-UV HB spectrum was measured by fixing the probe laser to each band represented by dotted lines.
agreement with the observed one, while others comparatively well reproduce the observed ones. To simulate the FC spectra by using the PGOPHER program, the maximum vibrational quantum numbers of each mode in the excited state should be set manually. There is thus a possibility of missing for that setting in the simulation of conformer B. We thus also calculated the FC spectra by using the PGOPHER program. Geometrical optimization and vibrational analysis prior to the FC simulation were carried out at the same level as that of Féraud et al. Fig. 7 also shows the newly calculated FC spectra of the four conformers. The frequencies of the lowest 10 modes are listed in
Table 1. By comparing the FC spectrum of conformer B of this work with the reported one [34], it was found that the latter does not include modes 3 and 5. In conformers A and C, the 21 transition appears, while not it but the 31 transition appears in conformers B and D, which can explain the difference between the two groups, (A, C) and (B, D). In the FC spectra of A and C, the intensity pattern of 11 and 21 is different, i.e., the former is stronger than the latter in A, while they are comparative in C. Since the UV-UV HB spectra are saturated, the intensity patterns are also calibrated from the UV photo-dissociation spectrum. From these spectra, it is found that the 11 transition of conformer A is stronger than 21. Thus,
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S.-i. Ishiuchi et al. / Journal of Molecular Spectroscopy xxx (2016) xxx–xxx
A 00 11 21
3
1251, 2171 1151 4151 13 1231 1 1 1 7 5 1 71 1 1 81 1131 1 4 1241 61 1331 1161
1121 2 41 1 1
C 00 11 2
1
1 1
12 12 22 41
1151 1 1 1 1 1 2 6 1 112141 7 1 6 1 1 8 1 1 1171 2 7 2 5 1 6
1141 1122 2141 51 1221
B 00
61 11
31
41 12
1141 314
1 1
13
1151 1 1 71 1 6
1 1 5
4151
1171
4161
D 00 1
11
0
50
3
1
51 6
41
81 416 1 4171
11511161 1171 4151
1141
100
71
150
200
250
Wavenumber / cm-1 Fig. 7. UV-UV HB spectra of the four conformers aligned by 0–0 transition energy and calculated FC spectra of each conformer.
Table 1 Vibrational frequencies (cm1) of the lowest 10 modes of each conformer in the S1 (pp⁄) state calculated at RI-CC2/aug-cc-pVDZ level. Mode
Conf. A
Conf. B
Conf. C
Conf. D
1 2 3 4 5 6 7 8 9 10
44.2 47.3 80.3 97.9 167.2 198.1 208.1 279.9 286.8 322.5
44.7 52.2 64.2 88.2 165.4 171.1 200.8 282.3 286.1 317.8
43.6 47.4 81.0 102.0 161.3 187.5 206.4 277.1 285.7 318.8
41.1 51.1 65.7 87.7 160.5 169.1 189.4 279.0 285.9 308.4
the structures of conformers A and C are determined as shown in Fig. 7. In the observed spectra of B and D, a difference of the 11 transition energy is found between them. That of B is 00 + 44.1 cm1, while it is 40.3 cm1 for D. This tendency is well reproduced by the calculation (see Table 1). Thus, the absolute OH orientation in the (B, D) pair is concluded to be shown in Fig. 7. 5. Conclusion To decrease the temperature of ions trapped in the QIT, we made it from copper. When a normal QIT made from stainlesssteel was employed, the temperature of TyrH+ was estimated to be 45–50 K, based on the rotational contour and the hot band intensity observed in the UV photo-dissociation spectrum of TyrH+. By replacing it by a copper QIT, the intensity of the hot bands observed in the UV spectrum drastically decreased. The temperature of the TyrH+ trapped in the copper QIT was estimated to be
13 K. So far, the reason for the low cooling efficiency by the cryogenic QIT has been thought to be due to RF heating, because it does not have a free field region, except for the center point. However, the result of this work clearly shows that the main reason for it is the material of the QIT. Interestingly, the temperature of the copper QIT, itself, was the same as that of the stainless-steel QIT. Nonetheless, the temperature of ions trapped in the former was much lower than that of the latter. This result indicates that the temperature of the buffer gas is different between them, which can be explained by the difference in the thermal conductivity, i.e., the thermal conductivity of stainless-steel is much lower than that of copper at the ultra-cold temperature, and that the low thermal conductivity causes a low cooling efficiency of the buffer gas. The cooling efficiency of cryogenic QIT can be improved significantly by making it from copper, which enables us to obtain conformer-selected UV spectra of cold ions by using UV-UV HB spectroscopy under a sufficiently cold condition. Its advantage was demonstrated by the UV-UV HB spectra of TyrH+. Four different UV-UV HB spectra were measured and the co-existence of four conformers was confirmed spectroscopically. From the spectral similarity, it was confirmed that conformers A and C, and conformers B and D are the phenolic OH rotamer pair, respectively. By comparing with the calculated FC spectra, the absolute phenolic OH orientations have been determined. Acknowledgements We are grateful for Prof. Gilles Grégoire, Prof. Yoshiya Inokuchi and Prof. Nam Joon Kim for giving us fruitful advice and inspiration to improve the cooling efficiency of the QIT. Mr. Kunihiro Aoshima and Mr. Hiroyuki Miyamoto at ADCAP VACUUM TECHNOLOGY CO, LTD (http://www.adcap-vacuum.com) provided help to us to make
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the copper QIT. We deeply thank them. This work was supported in part by a Grant-in-Aid for Scientific Research KAKENHI on Innovative Area (2503): ‘‘Studying the Function of Soft Molecular Systems by the Concerted Use of Theory and Experiment’’, KAKENHI in the priority area ‘‘Molecular Science for Supra Functional Systems’’, a Grant-in-Aid for Young Scientists KAKENHI, and the Cooperative Research Program of ‘‘Network Joint Research Center for Materials and Devices’’, from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan.
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