Dependence of Adenine Raman Spectrum on Excitation Laser

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Dependence of Adenine Raman Spectrum on Excitation Laser Wavelength: Comparison between Experiment and Theoretical Simulations Navchtsetseg Nergui, Miin-Jang Chen, Juen-Kai Wang, Yuh-Lin Wang, Cheng-Rong Hsing, Ching-Ming Wei, and Kaito Takahashi J. Phys. Chem. A, Just Accepted Manuscript • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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The Journal of Physical Chemistry

Dependence of Adenine Raman Spectrum on Excitation Laser Wavelength: Comparison between Experiment and Theoretical Simulations Navchtsetseg Nergui,abc Miin-Jang Chen,d Juen-Kai Wang,ae Yuh-Lin Wang,af Cheng-Rong Hsing,a Ching-Ming Weia and Kaito Takahashi*a a

Institute of Atomic and Molecular Sciences, Academia Sinica, PO Box 23-166, 10617 Taipei, Taiwan

b

Department of Chemistry, National Taiwan University, 10617 Taipei, Taiwan

c

Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica and National Taiwan University d e f

Department of Materials Science and Engineering, National Taiwan University, 10617 Taipei, Taiwan

Center for Condensed Matter Sciences, National Taiwan University, 10617 Taipei, Taiwan

Department of Physics, National Taiwan University, 10617 Taipei, Taiwan

*Corresponding author e-mail: [email protected]

ABSTRACT We acquired the Raman spectra of adenine in powder and aqueous phase using excitation lasers with 532, 633 and 785 nm wavelengths for the region between 300 to 1500 cm-1. In comparison to the most distinct peak at 722 cm-1, the peaks between 1200 and 1500 cm-1 exhibited a characteristic increase in cross-section with decreasing excitation wavelength in both phases. This trend can be reproduced by different density functional theory (DFT) calculations for the adenine molecule in the gas phase as well as in the aqueous phase. Furthermore, from the calculation on the -stacked dimer, hydrogen bonded dimer and trimer, we find that this trend towards excitation laser wavelength is not sensitive to the packing. When comparing the Raman spectra given by different excitation wavelength, care should be taken in analyzing the cross-section and present day DFT calculations are able to capture general trends in the excitation laser wavelength dependence of the Raman activity.

INTRODUCTION Adenine is considered to be one of the most important biomolecules on Earth because it is a nucleobase responsible for genetic transcription/translation and a key building block for molecules such as adenosine triphosphate that is the energy source of living organisms.1 Over the years, many analytical 1

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methods have been employed to study its molecular structure as well as chemical properties. Among them, Raman spectroscopy has been used to measure its vibrational spectrum and most of its vibrational peaks has been assigned.2–10 Furthermore, studies on pre-resonance and resonance Raman spectra with excitation laser wavelengths of 210 to 340 nm have helped understand variations in the electronic excited state of adenine derivatives.11–15 In a recent surface-enhanced Raman study of adenine on Rh electrode surface, Cui et al. noticed that the peak at 1321 cm-1 becomes stronger as the excitation laser wavelength decreases from 632.8 nm to 325 nm.10 They attributed this to pre-resonance effects. However, considering that the strong electronic transitions for adenine are observed at 250 and 200 nm,16–22 it is very curious that such effects can be observed in the visible excitation laser wavelengths. Therefore in the present study, we performed systematic experimental analysis on the peaks in the 300 to 1500 cm-1 range for adenine using lasers in the visible range. Our understanding of adenine Raman spectra can be further deepened from the comparison between experimental and theoretical peak positions as well as Raman activities. The different theories for Raman activities developed in the 70’s allow us to connect pre-resonance Raman activities to corresponding electronic transitions.23–28 Furthermore, due to the enormous acceleration in the computing power and rapid development in quantum chemistry computation techniques in the last two decades, various density functional theory (DFT) based methods are available for the calculation of Raman activities at different excitation laser wavelengths.29–35 In addition, calculations on the electronic excited states can be used to understand the trends of the Raman activities.36 In the present study, we performed a systematic comparison between experimental and theoretical Raman spectra for adenine excited by three laser wavelengths: 532, 633 and 785 nm. The experimental studies were performed for powder and aqueous phase adenine. This molecule has a strong set of signature peaks in the range of 300 to 1500 cm-1. We selected 9 peaks in this region and analyzed the variation in the Raman intensities as a function of excitation laser wavelength. We analyzed these experimental results using many different DFT functionals that are utilized in Raman studies to gauge the accuracy of different methods. In addition, to study the effects of clustering we performed calculations for adenine monomer, the -stacked dimer, and the hydrogen bonded dimer and trimer sheets. There have been many studies comparing the experimental and calculated peak positions of adenine Raman spectra.5–8 However, to the best of our knowledge, we did not find comparisons concerning the excitation laser wavelength dependence of the Raman activity. In this paper, we tested several DFT functionals to judge whether they can reproduce the experimental excitation laser wavelength dependence. Furthermore, there are many studies on pre-resonance Raman11–14 using the excitations in 210 to 340 nm which are analyzed based on formulas given by Albrecht and coworkers25,26 as well as by Ting27. These equations, which are based on the sum of state formulation of the excitation laser wavelength dependent polarizability, are also used to analyze the obtained trends in the Raman activity.

2


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METHODS A. Experimental Methods. Adenine powder was purchased from Sigma-Aldrich with 98.9% purity and used without further purification. A saturated aqueous solution of adenine was prepared by adding small amounts of adenine powder to water and stirring until the powder dissolved. We repeated this until precipitation was observed at the bottom of the beaker. Then we took the solution at top of the beaker, and filtered it before measuring the Raman spectra. The Raman experiments were performed in a Raman microscope (HR800, Horiba). The excitation lasers were emitted at three different wavelengths: frequency-doubled Nd:YAG laser emitting at 523 nm, HeNe laser emitting at 632.8 nm, and single-mode semiconductor diode laser emitting at 785 nm. The laser beam, after passing through a corresponding laser-line filter to remove residual plasma lines or amplified spontaneous emission, was focused by a 10 micro-objective lens to the adenine powder (or adenine aqueous solution) placed on a glass slide. The irradiated laser power was measured for each excitation laser. The scattered radiation was then collected by the same micro-objective lens in backward direction and passed through a long-pass filter to remove the residual laser radiation. The collected radiation was then sent to an 80-cm spectrometer plus liquid-nitrogen cooled charge-coupled device (CCD). The spectral resolution and error are 7 and 0.1 cm-1, respectively. To compare the signal strengths of the Raman spectra acquired with the three excitation lasers, a spectral intensity calibration procedure was taken to account for the different wavelength dependences in the optical throughputs of the Raman system (including the micro-objective lens, filters, and the spectrometer) and in the quantum efficiency of the CCD, as indicated by the expression of the measured anti-Stokes Raman spectrum (in unit of count sec-1) below: , where

(1)

is the wavelength corresponding to specific Raman peak at the frequency of

in unit of wavenumber (cm-1), wavelength

(in unit of watt),

is a constant,

is the laser incident intensity at excitation

accounts for the integrated wavelength dependence of the optical

throughputs of lens, mirrors, and filters,

is the wavelength dependence of the diffraction

efficiency of the grating in the spectrometer at a

-dependent rotational angle

wavelength dependence of the quantum efficiency of the CCD, and finally dependence of Raman cross-section with excitation laser wavelength

,

is the

is the frequency

. Eq 1 is derived from eq 2.11

of Ref. 38 with the consideration of the wavelength dependences of the Raman system. A calibration procedure was taken to extract . First, the output of a deuterium-halogen light source with calibrated spectral output (DH-2000, Ocean Optics) was sent to the optical path of the collected radiation (from the micro-objective lens, through the low-pass filter and the 80-cm spectrometer, and to the CCD). We present the spectral irradiance of the calibrated lamp in Figure S1 in Supporting 3



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Information (SI). The intensity spectra of the calibrated lamp with the grating orientations adopted at the three excitation wavelengths are given below: , where

is a constant and

Based on eqs 1 and 2,

is the intensity spectrum of the calibrated lamp (in unit of W nm-1). is expressed as .

Namely, the calibrated Raman spectrum and

(2)

calculated from the measured

(3) ,

,

can be used as the experimental cross-section. We then determined the peak

position and Raman cross-section by fitting the peaks to a Gaussian function. The area of the Gaussian of a specific Raman peak at . The function is used as the integrated cross-section experimental conditions for the powder and aqueous phase adenine are the same, thereby the above procedure is applicable to both phases. Lastly, we also note that another possible source to perform spectral intensity calibration is the laser-generated broadband luminescence from a calibrated luminescent material.37,38 In this study, we nevertheless used the white-light calibrated lamp. In Figure S2 of SI, we present the calibration procedure of the Raman spectra for powder adenine obtained with the 532 nm excitation laser. B. Computational Methods. We performed optimization and vibrational calculation for the singlet ground electronic states of adenine using B3LYP,39,40 B3PW91,41,42 and M06-2X43 functional with the 6311+G(d,p)44,45 basis set. To test the basis set dependence, we also performed B3LYP calculation with Dunning’s aug-cc-pVTZ46–48 basis set. To model the crystal packing geometry, we further performed B3LYP-D249/6-311+G(d,p) calculation for the adenine dimer and trimer. To study the modifications to the calculation results by different solvation models, we performed simulations using B3LYP/6311+G(d,p) with the solvent model based on density of Truhlar and coworkers.50 To test the sensitivity of the results on the solvent, we tested for water, acetone, and benzene. We performed the Raman simulation at the three excitation wavelength used in the experiment as well as at 488 nm. All calculations were performed using the Gaussian 09 program.51 Schematic plots of the adenine molecule and clusters considered in the present study are given in Figure 1. XYZ geometries of the calculated adenine molecule and clusters are given in SI. Adenine molecule can have many isomers, however previous studies have shown that at room temperature, only one isomer, N9H, is present.4,6 Previously, it has been reported that the crystal packing geometry of adenine is obtained by the hydrogen bonding of the amine group NH bond and the Nitrogen atom in the two rings, as well as by the π-stacking interaction.6 For the -stacked dimer, we selected the most stable conformer reported by Bravaya et al.52 For the dimer (trimer) we report the average peak position and Raman activity of the 2 (3) corresponding vibrational modes. 4


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The Raman cross-section for the j-th normal vibrational mode was calculated by using the following equation29: , where

and

the fundamental wavenumber

(4)

is the Raman activity of the j-th vibrational normal mode at , respectively; and h, c and kB are Planck constant, light speed and

Boltzmann constant, respectively. We note here that this equation is based on the assumption that the scattered light is averaged by the different rotational angles of the molecule.29 Therefore, this equation is not valid if the sample is a neat single crystal with a definite orientation with respect to the directions of the incident laser and the radiation collection. Since the sample used in this work was in powder form consisting of randomly oriented fine crystals or in aqueous phase, the obtained Raman data would make good correspondence to the simulated results.

Figure 1. Schematic plots of adenine (a) monomer, (b) hydrogen bonded dimer, (c) -stacked dimer, and (d) hydrogen bonded trimer calculated using B3LYP-D2/6-311+G(d,p).

The Raman activity

is proportional to the squared value of the normal mode derivative of

the excitation laser wavelength dependent polarizability ,

(5)

5



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where

and

are Cartesian directions (X, Y, or Z) and

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is the jth normal mode coordinate. From the

sum over state formation for the frequency dependent polarizability, the normal mode derivative can be given as29 , where the sum is for all excited electronic states k, electronic state, and

and

(6)

are the energy and wavefunction of the kth

represents the transition dipole moment in the

direction between the

ground and kth electronic state. In the pre-resonant case, one excited state

is accessed with the

excitation photons—namely,

, and the summation in eq 6 is dominated by the

contribution of this resonant excitation. Eq 6 can be approximated as .

(7)

We note that in the calculation used in the Gaussian09 program, this sum of state is avoided by using the quasi-energy formulation.30–35

RESULTS AND DISCUSSION A. Comparison on Vibrational Frequency. The normalized Raman spectra of adenine powder at three excitation wavelengths are shown in Figure 2. As can be seen from this figure, the peak positions do not change with the excitation wavelength, but the relative intensities of the peaks in the 1200-1500 cm-1 region greatly decrease as the excitation wavelength varies from 532 nm to 785 nm. The 532 nm spectra are consistent with the previous experimental spectra by Lopes et al. on anhydrous adenine obtained at 514 nm excitation wavelength.6 We note that peak positions reported by Lopes et al. are systematically blue-shifted by 2 cm-1 compared with our results. Such discrepancy could be the result of spectral calibration. Table 1 lists the peak positions of the 9 peaks selected for the present analysis which are consistent with previous literatures.6–8 The most prominent peak at 722 cm-1 can be assigned to the ring breathing motion and is seen in the Raman spectra of most purine derivatives. The schematics of the normal mode vibrations considered in the present study are given in Figures S6-S9 of SI. In Figure 3, we present the experimental spectra of the aqueous phase adenine, and the peak positions obtained from the 532 nm spectra are given in Table 1.

6


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Figure 2. Experimental Raman spectra of adenine powder at three excitation wavelengths: 532 nm (green), 633 nm (red), and 785 nm (brown). The peaks in each spectrum are normalized to the one at 722 cm-1. Nine peaks studied in the present paper are marked with arrows.

Figure 3. Experimental Raman spectra of aqueous adenine solution at three excitation wavelengths: 532 (green), 632.8 (red), and 785 nm (blue). The peaks in each spectrum are normalized to the one at 722 cm-1.

Table 1. Peak position in cm-1 of the 9 peaks in the experimental and calculated spectra. The calculated results of adenine monomer were obtained with B3LYP/6-311+G(d,p) and B3LYP-D2/6-311+G(d,p), 7



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and that of two adenine dimer configurations (H-bond and

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-stacked dimer) and H-bond trimer

configuration were obtained with B3LYP-D2/6-311+G(d,p). Previous literature values from reference 5 and 6 are also presented. Peak

1

2

3

4

5

6

7

8

9

Powder Experiment

534

621

722

940

1023

1125

1247

1332

1482

Aqueous Experiment

533

621

722

946

1019

1120

1250

1332

1484

B3LYP monomer

520

618

725

945

1008

1079

1242

1356

1513

B3LYP-D2 monomer

522

618

724

942

1014

1068

1242

1354

1511

B3LYP-D2 H-bond dimer

534

629

727

951

1043

1076

1261

1356

1517

B3LYP-D2 -stacked dimer

528

621

726

944

1041

1074

1255

1355

1515

B3LYP-D2 H-bond trimer

536

637

728

949

1056

1073

1264

1356

1505

Aqueous results ref 5

529

616

720

949

1027

1144

1242

1352

1497

Powder results ref 6

537

623

724

943

1025

1127

1250

1334

1484

From the comparison of the different calculation results shown in Table 1 and in Table S1 of SI, the Raman peaks below 1000 cm-1 are within a few cm-1 of the experimental values without any scaling. On the other hand, those above 1000 cm-1 are off by ~30 cm-1. Given this good correspondence, the subsequent analysis is done without any spectral scaling. In Table S2 of SI we list the root mean square error of the different DFT methods, for monomer and dimer, and for monomer in three solvents (water, acetone and benzene). We find that: (1) B3LYP and B3LYP-D2 methods give spectral error of ~20 cm-1, while B3PW91 and M06-2X confer slightly larger error; (2) the spectral error is similar for the monomer and dimer calculation, signifying that the vibrational modes of interest here are not sensitive to the molecular clustering; (3) the peak position errors of monomer in gas phase and in three solvents (water, acetone and benzene) are also alike, indicating that the spectral positions of the peaks between 300 to 1500 cm-1 are also unaffected by solvation. These results give us confidence in analyzing the Raman activity between the calculated and experimental results for the region between 300 to 1500 cm-1. We note that if one looks at lower wavenumbers for the powder case, the van der Waals stretching modes which are absent in the monomer calculation will show up. At larger wavenumbers, the NH bending and NH stretching peaks will also show shifts due to the hydrogen bonding interaction which are absent in the monomer calculation. B. Comparison on Raman Activities. If one observes the experimental Raman spectra given in Figures 2 and 3, one can clearly notice that compared to the sharp peak at 722 cm-1, the peaks above 1200 cm-1 increase at shorter excitation 8


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wavelengths. For the powder spectra, in Figure 2, we are able to obtain quantitative values for the integrated cross-section at all excitation laser wavelengths. On the other hand, for the aqueous spectra, in Figure 3, it is hard to obtain quantitative results for the excitation wavelength dependence due to the large fluctuation in the 785 nm data. In Figure 4, we present the calculated spectra of the gas phase adenine molecule obtained by B3LYP/6-311+G(d,p). Though not as obvious as the experimental results, one can see that the peak at 1332 cm-1 increase at shorter excitation laser wavelengths.

Figure 4. The calculated Raman spectra of adenine molecule obtained with B3LYP/6-311+G(d,p) at three excitation wavelengths: 532 nm (green), 633 nm (red), and 785 nm (brown). All the peak intensities in each spectrum are normalized to the one at 725 cm-1. To facilitate the comparison in Raman activity, the experimentally acquired integrated cross-section of each Raman peak was first converted to their corresponding Raman activity:29 .

(8)

Both the experimental and calculated Raman activities are then normalized to that of the ring breathing mode of adenine at ~722 cm-1: .

(9)

Finally, the normalized Raman activities at the excitation wavelengths of 532 and 633 nm are divided by 785 nm: that at

9



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.

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(10)

Therefore, if the normalized Raman activity has the same excitation wavelength dependence as the 722 cm-1 peak,

. Figure 5 shows

at 722 cm-1. Notice that photon energy,

and

and

of the 8 Raman peaks, except the one

of peaks 7, 8, and 9 increase conspicuously with the excitation

. On the other hand, the corresponding values of the low-frequency peaks 1 and 4

exhibit milder increase with respect to

. We also note that other than Peak 1, the

peaks all increase more dramatically with

compared to the increase of

of the other . Such excitation

wavelength dependence does not depend on whether the calculation is for adenine monomer, dimer, or trimer, indicating that this behavior is insensitive to molecular clustering for the peaks studied in the present range of 300 to 1500 cm-1. Here it is very interesting that different vibrational modes show different excitation laser wavelength dependence, even though the lasers used in this study are all far from being resonant.

10


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1.15

1.60

B3LYP-D2 Monomer B3LYP-D2 Hbond Dimer B3LYP-D2 -stacked Dimer B3LYP-D2 trimer Exp

1.10

1.45

1.05

1.30

1.00

1.15

0.95 12000

15000

λ (cm (a) peak 1 ex-1

18000

21000

1.00 12000

1.30

1.60

1.23

1.45

1.15

1.30

1.08

1.15

1.00 12000

15000

λ (cm ) (c) peak 4 ex-1

18000

21000

1.00 12000

2.00

1.45

1.75

1.30

1.50

1.15

1.25

15000

λex-1 (cm-1) (e) peak 6

18000

21000

1.00 12000

2.00

2.00

1.75

1.75

1.50

1.50

1.25

1.25

1.00 12000

15000

18000

21000

15000

18000

21000

15000

18000

21000

15000

18000

21000

λex-1 (cm-1) (b) peak 2

λex-1 (cm-1) (d) peak 5

-1

1.60

1.00 12000

15000

-1)

λex-1 (cm-1) (g) peak 8

18000

Figure 5. Normalized Raman activity ratios,

21000

1.00 12000

and

λex-1 (cm-1) (f) peak 7

λex-1 (cm-1) (h) peak 9

, versus excitation wavenumber,

, for the

8 peaks: (a) Peak 1, (b) Peak 2, (c) Peak 4, (d) Peak 5, (e) Peak 6, (f) Peak 7, (g) Peak 8, and (h) Peak 9. Cross symbols are experimental data; red circles are calculated data by B3LYP-D2 monomer; purple 11



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diamonds are calculated data by B3LYP-D2 hydrogen bond dimer; green squares are calculated data by B3LYP-D2 -stacked dimer; blue triangles are calculated data by B3LYP-D2 trimer.

Considering the similarities seen between the Raman activity ratios of the calculated and experimental results, we can perform further analysis using the calculated results. Using the Raman activities obtained from the Gaussian 09 program, we may take the ratio of the Raman activity of each vibrational mode versus that of the 785 nm laser. , where

(11)

. As mentioned in the method section, if the Raman activity is dominated by a

single electronic transition to the

-th state, in the pre-resonance case, the Raman activity can be given

as .

(12)

Next, if this pre-resonance condition is valid, the Raman activity ratio can be expressed as , where

(13)

. For near resonance conditions, Albrecht and Hutley26 as well as Ting27 have

also proposed equations which show similar excitation laser wavelength dependence as eq 13. For adenine molecule, it was experimentally shown that there are strong electron transitions at around 250 and 200 nm16–18. Using the time dependent (TD) variant of B3LYP/6-311+G(d,p), we calculated that gas phase adenine molecule has strong absorption at 248, 198, 186, 175, 167 and 148 nm— corresponding to 5.0, 6.3, 6.6, 7.1, 7.4 and 8.3 eV, respectively, which are consistent with the calculation numbers reported by Barbatti et al. using more accurate methods.22,53 Cluster formation or solvation does not greatly change these values. Figure 6 shows the calculated Raman activity ratios of the 9 Raman peaks, , along with those obtained from eq 13 using 5.0, 6.3 and 7.4 eV excitation. One can notice groupings of the peaks, for example peaks 7, 8, 9 show large excitation laser wavelength dependence, while peaks 1, 3, 4 show small dependence, and peaks 2, 5, 6 lie in between. Another interesting feature is that peaks 1, 3, and 4 follow the excitation laser wavelength dependence of eq 13 with =6.3 eV, while peaks 2, 5, 6 follow that of =5.0 eV. From this plot it seems that the Raman activity of certain vibrational modes may be correlated to a specific electronic transition. 12


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2.5 2.2

peak 1 peak 4 peak 7 eq 13 5.0eV



1.9 1.6 1.3 1.0 12000

Figure 6. Raman activity ratio

15000

λex-1 (cm-1)

18000

21000

of adenine molecule of 9 Raman peaks (see Table 1) obtained with obtained by eq 13 for the three strong lowest energy

B3LYP/6-311+G(d,p), as well as

electronic transition of adenine molecule at 5.0 (dotted curve), 6.3 (dashed curve) and 7.4 eV (solid curve) versus excitation wavenumber, . To test this hypothesis, we used the sum of state equation given above, eq 6, and numerically determined the contributions of each electronic state for the vibrational normal modes responsible for peaks 1 to 9. Using TD-B3LYP/6-311+G(d,p) for the adenine molecule, we calculated the electronic excited state energies and the transition moments to 99 excited electronic states. We note that TDB3LYP lacks the ability to describe Rydberg excitations54, charge transfer excitations55, and double excitations56,57, therefore the accuracy of the highly excited electronic states in the sum of state equation is questionable. However, the main goal here is to understand if the Raman activities of a given vibrational mode can be correlated to a specific electronic transition, and not to obtain exact agreement with experimental results. Indeed, it was seen in Fig. 5, that the TD-B3LYP results slightly underestimate the input laser wavelength dependence compared to the experimental results. Here we confirm the accuracy of the TD-B3LYP results for low lying excitations by comparing to the more accurate multi-state complete active space second order perturbation calculations using the aug-ccpVTZ basis given by Barbatti et al.53 For the dark n-π* transition, they report a peak at 5.04 eV with an oscillator strength of 0.002, while TD-B3LYP gives a peak at 4.93 eV with 0.001 oscillator strength. For the low lying strong π-π* transition, they report a peak at 5.11 eV with 0.398 oscillator strength while we obtain 4.99 eV with 0.205. Lastly, in the weakπ-π* transition, their values are 5.02 eV with 0.015 oscillator strength, while we get 5.25 eV with 0.042. As conclusion, the present TD-B3LYP calculation is accurate to a few tenth of an eV while the oscillator strengths are within a factor of three. This accuracy should give the correct trend, and we will use the sum of state equation to numerically obtain the normal mode derivative of the excitation wavelength dependent polarizability. Using the 99 excited electronic states obtained by TD-B3LYP, we performed numerical derivative with respect to the normal coordinate

and obtained the derivative term

. 13



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For the numerical derivation, we performed convergence test by using two values:

0.01 and 0.001

Å, confirmed that the variation in the obtained derivative is less than 5%. We note that Williams et al have calculated Raman activities using similar techniques for the frequency independent polarizability.58 For the adenine molecule we calculated the

,

,

,

since we are dealing

with in plane vibrations for the adenine molecule, where the Z axis is defined as the one out of the adenine plane. As given in Tables S3-S11 of SI, we find that for all 9 peaks studied here, the dominant contribution toward the polarization derivative is coming from the excitations to 6.3 and 7.4 eV. However, it is not possible to correlate the Raman activity of one vibrational mode to one specific electronic transition. Thereby we conclude that the excitation laser wavelength dependence of the Raman activity for the peaks studies in the present research show trends that seem to follow pre-resonant Raman conditions, but that does not mean that the Raman activity can be assigned as coming from a specific electronic excitation in the sum of state formulation. We note that Dudik et al. reached a similar conclusion that many excited electronic states contribute to the Raman activity.59 As seen in Tables S3S11 of SI, neighboring states give contributions of opposite signs which makes the analysis more complicating. More systematic studies on other purine derivatives are needed to understand the cause for the excitation laser wavelength behaviors we see in Figure 6.

CONCLUSIONS We studied the variation in Raman spectra at three different excitation laser excitation wavelength for adenine in the range of 300 to 1500 cm-1. From the comparison with experimental values, we found that the peak position and the Raman cross-section show small DFT functional dependence. Compared to the distinct 722 cm-1 peak, the cross-section of the peaks in the 1200 to 1500 cm-1 region decreased greatly in going from 532 nm to 785 nm. This result is consistent with the analysis given by Cui et al.10 concerning their excitation wavelength dependence on the surface enhanced Raman spectra of adenine on Rh electrode. All the DFT functionals studied in the present paper were able to reproduce this feature. In addition, the calculation results showed that hydrogen bonding and -stacking clusters as well as solvation does not change the general trend in the excitation laser wavelength dependence. We find that an equation based on pre-resonant condition, can be applied for the wavelength dependence in the present case, even though the excitation laser wavelength is very far from resonance. However, detailed studies using the sum of state equation for the excitation laser wavelength dependent polarizability show that such wavelength dependence is not exactly connected to the excitation to one specific excited electronic state. Further studies on the vibrational modes below 300 cm-1 or above 1500 cm-1, as well as studies with other excitation laser wavelengths such as 1015 or 488 nm are required to gain a general understanding of this excitation laser wavelength dependence. Studies on other purine derivatives should also help shed light to this interesting excitation laser wavelength dependence of the Raman activity.

14


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Acknowledgement KT thanks Academia Sinica, National Center for High Performance Computing and Ministry of Science and Technology (NSC 102-2113-M-001 -012 -MY3) of Taiwan for support. Supplementary Information (SI) available: The experimental detail, comparison of the powder and aqueous Raman spectra, comparison of the calculated peak position, Raman activity ratio versus excitation laser wavenumber for different calculation methods, schematic vibrational modes of the adenine hydrogen bonded dimer, adenine π-stacked dimer, and adenine hydrogen bonded trimer, the numerical results from the sum over state formula, as well as the XYZ geometry of the calculated structures are given in the supplementary information.

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Table of Contents:

532nm

Intensity, (AU)

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Page 20 of 20

633nm

785nm 200

400

600

800

1000

1200

1400

1600

Raman shift (cm-1)

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Dependence of Adenine Raman Spectrum on Excitation Laser

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