EtdBr consists of many functional groups such as amine, imine and hydrophobic ring groups. As an aromatic compound, luminescence is predictable for EtdBr.
Although EtdBr is emissive in organic solvent, the emission intensity is weak in aqueous solution. The quantum yield in aqueous solution is about one third of that in organic solvent.18 It was proposed that in aqueous solution the amine groups are subjected for proton quenching to eliminate the emissive excited state. But in other protic solvent proton quench is not obvious. Thus proton quench is not the cause and there is another argument.
Olmsted III and Kearns ascertained that the viscosity of the medium is irrelevant to the emissive property of EtdBr as the emission quantum yield and fluorescence lifetime are very similar in methanol and glycerol, whose viscosities are 0.55 cP and 954 cP; respectively.18 They also concluded that proton transfer from the excited state is the principal process responsible for the low emission quantum yield in most polar protic solvents from the results of fluorescence quenching by proton acceptors and the substantial extended lifetime observed upon deuteration of the amine groups on EtdBr, regardless of the medium. Therefore, the stronger the proton acceptor ability of the solvent, the lower the emission intensity is observed. The excited state proton transfer from the imine groups in the quinonoid form of ethidium cation (structure II in Figure 25) to solvent is proposed as the mechanism. The imino proton becomes quite acidic in the ground state, and is expected to be more acidic in the excited state, similar to other aromatic amines.
Pal and colleagues had also suggested that the fluorescence quench of EtdBr should depend on the hydrogen-bond acceptor ability of the solvent ( instead of the polarity such as dielectric constant or molar transition energy.29 The quenching rate
constants by water and hydroxide ion in mixed acetonitrile-water and bulk water solution are 1.7 x 107 M−1s−1 and 4.4 x 1010 M−1s−1; respectively. They concluded that the quenching ability of hydroxide ion is nearly 2500 times and more efficient
compared to water. This result is consistent with the proton transfer model, proposed earlier by Olmsted III and Kearns. The fluorescence quenching by hydroxide ion in aqueous solution may be attributed to abstraction of the imino proton in ethidium cation (structure II in Figure 24). Once happened the process is irreversible and the deprotonated ethidium cation is non-emissive.
Figure 25. Resonance structures of ethidium cation.
Both the proton and hydrogen-bond acceptor ability of the solvent have relations with the basicity of the solvent. In this study, the photophysical properties of EtdBr in various pH values have been studied both in aqueous (Figure S5 and S6) and
non-aqueous solution (Figure S7−S10). In the NaPi buffer solution the absorption maximum and emission intensity are nearly the same between pH 3 to pH 10 (Figure 26). Similarly, the absorption maximum and emission intensity in CH3CN remain constant between pH 2 to pH 9 (Figure 27). Only at extreme pH values (below pH 2, above pH 10), the emission intensity starts to decline. However, it can be indicated from Figure 26 and 27, the emission intensity remains constant in broad ranges of pH values. Consequently, solvent basicity is clearly not the cause to deactivate the emissive excited state.
0 2 4 6 8 10 12 14
Figure 26. Emission intensity at 615 nm (excited at 460 nm) and absorption maximum of n→* transition for EtdBr between pH 0−14 in NaPi buffer solution.
0 1 2 3 4 5 6 7 8 9 10 11
Figure 27. Emission intensity at 610 nm (excited at 510 nm) and absorption maximum of n→* transition for EtdBr between pH 1−10 in CH3CN. The emission intensities are calibrated by the absorbance at 510 nm for pH 1−9. At pH 11 and 12 the emission intensities are not shown due to the absent of n→* transition.
Besides, it is noteworthy that in the extremely basic condition the absorption spectra of EtdBr are different in aqueous and non-aqueous solution. In the extremely basic NaPi buffer solution, the n→* transition band is nearly unchanged, while in CH3CN it disappears. Simulation of the electronic transition for ethidium cation and deprotonated ethidium cation is exploited to explain the above phenomenon. The deprotonated ethidium cation has two structures due to the asymmetry of the two amine groups. For conveniently the two deprotonated ethidium cations are simplified to Etd-RH and Etd-LH which represent extracting proton from the right form amine and left form amine; respectively. The calculated n→* transition for ethidium cation has maximum absorbance at 526 nm which is rather red shift compare to the
experimental data (Figure 27). The calculated n→* transition for Etd-RH and Etd-LH are further red shift to 668 nm and 835 nm; respectively (Figure 29 and 30).
After deprotonation more electron delocalization from the negatively charged nitrogen of the amine group may cause the red shift of absorption spectra. Combining the theoretical calculations with the experimental data we propose that there is partial deprotonation in extremely basic NaPi buffer solution, while the deprotonation is completed in CH3CN. In extremely basic organic solvent, ethidium cation is completely deprotonated accompanied with the disappearance of absorption band which results no emission. In extremely basic aqueous solution, ethidium cation is partial deprotonation due to the surrounding of water molecules and the ethidium cation and deprotonated ethidium cation are in the fast equilibrium in the ground state and excited state. In the ground state the protonated form is dominated hence the absorption band is constant. However, in the electron-rich * excited state, the deprotonated form is dominated so that emission intensity decreases.
Figure 28. UV−Visible absorption spectrum of ethidium cation with predicted TD-DFT transition.
Figure 29. UV−Visible absorption spectrum of deprotonated ethidium cation (Etd-RH) with predicted TD-DFT transition.
Figure 30. UV−Visible absorption spectrum of deprotonated ethidium cation (Etd-LH) with predicted TD-DFT transition.
To deeply interpret the evolvement of fluorescence spectra for EtdBr in SDS solution, we propose two non-radiative decay pathways: intrinsic decay, (knr,I) and proton quench decay, (knr,H). EtdBr has weak emission in the aqueous solution due to existence of the knr,I and knr,H. Moreover, the emission intensity is further lower when SDS concentration increases up to 0.1%. If any hydrophobic interaction between SDS and EtdBr involves, it will reduce the value of knr,H which results increased emission intensity. Therefore, hydrophobic interaction is not the cause here. Due to the red shift of the absorption spectra, decreased emission intensity is rationale to the energy gap law.41,42 The lower energy of the excited state has better vibrational overlap with the ground state which facilitates knr,I of the excited state and leads to lower emission intensity. To verify this assumption, time-correlated single photon counting (TC-SPC) had been utilized to measure the fluorescence lifetime of EtdBr in various
concentrations of SDS solution (Figure 31 and S11−17). At 0−0.1% of SDS, the fluorescence lifetime of EtdBr ranges from 1.7 ns to 1.93 ns which are roughly the same, while above 0.1% SDS, the lifetime lengthen (Table 2). The slight difference of the lifetime below 0.1% SDS is within the uncertainty of the instrument. The result excludes the energy gap law since the fluorescence lifetime does not decline simultaneously below 0.1% of SDS. Comparing the emission intensity ratio with fluorescence lifetime ratio below 0.1% of SDS makes the variation more significant (Figure 32).
10 15 20 25 30 35
0 100 200
C o u n ts
Time, ns
0.1%, 1.93 ns IRF
fit
Figure 31. Luminescence decay of EtdBr in 0.1% of SDS. Black curve: experimental data, excited at 480 nm and received at 615 nm. Red curve: curve fitting of
experimental data. Blue curve: instrumental response function.
Table 2. Fluorescence lifetime and normalized emission intensity of EtdBr in various concentrations of SDS.
[SDS], % 0 0.001 0.005 0.01 0.05 0.1 0.2 1
, ns
1.7 1.69 1.73 1.86 1.79 1.93 3.19 4.87 I/Ioa 1 0.98 0.79 0.46 0.16 0.12 1.7 2.88aIo is the emission intensity of EtdBr in pure water.
0.00 0.05 0.10
0.5 1.0
I I
oSDS %
0.5 1.0
Figure 32. Emission intensity ratio at 623 nm (excited at 491 nm) and fluorescence lifetime ratio at 615 nm (excited at 480 nm) for EtdBr below 0.1% of SDS. Io and o
are the emission intensity and fluorescence lifetime in pure water; respectively.
On the other hand, red shift of absorption spectra indicates the complex formation between EtdBr and SDS. EtdBr salt bridged with SDS results in static quenching of the excited state because EtdBr-SDS complex is regard as non-emissive compound. Static quench means that luminophor (EtdBr) and quencher (SDS) form a stable complex in the ground state before excitation occurs. The remaining free unbinding EtdBr exhibits the same lifetime as in pure water. The observed decrease of emission intensity and similar emission profile are in accordance to the smaller
amounts of unbinding EtdBr. Figure 32 clearly demonstrates that the emission intensity decreases and the fluorescence lifetime is constant as SDS concentration increases from 0% to 0.1%.
At above 0.1% of SDS, aggregation of SDS to form micelle is in progress. The compact structure of SDS micelle protects EtdBr from the attack of water molecules and inhibits knr,H of the excited state. Therefore, the emission intensity increases gradually as the SDS micelle starts to form. After the SDS concentration increases above CMC the emission intensity reaches plateau due to the complete formation of SDS micelle. The emission intensity exceeds the initial value in pure water where knr,I
and knr,H both effect. Interestingly the normalized emission intensity and fluorescence lifetime are nearly the same above 0.1% of SDS (Figure 33).
0.0 0.2 0.4 0.6 0.8 1.0
0 1 2 3
I I
oSDS %
0 1 2 3
Figure 33. Emission intensity ratio at 623 nm (excited at 491 nm) and fluorescence lifetime ratio at 615 nm (excited at 480 nm) for EtdBr below 1% of SDS. Io and o are the emission intensity and fluorescence lifetime in pure water; respectively.