The photophysical data ofBkBandBkRare given in table 11.
Table 11:Photophysical properties of bichromophores with parallel chromophores in CHCl3.
Compound λmaxabs /nm (ε/m-1cm-1) λmaxem /nm Φf τf/ns BkB(116) 357 (93,000) 394 0.38a 1.03±0.05 BkR(118) 548, 358 (n.d.)b 618 n.d. 2.6±0.1 (a) Fl. quantum yieldΦf was measured using Coumarin 120 in MeOH (Φf=0.51) as a reference;[132](b)εnot determined, ratio of absorbance is 1.00:0.81.
Figure 48 shows the normalized absorption/emission spectra of bichromophores with identical or different chromophores and their combinations with parallel or perpendicular orientations, respectively. As references, the spectra of "blue"
and "red" model compoundsBandR, are shown in figure 48a. In figure 48b, the spectra of bichromophores with two identical chromophores (B⊥B, BkB) are shown, while spectra of bichromophores with different chromophores (B⊥R, BkR) are shown in figure 48c. Bichromophores with red chromopohores R⊥R andRkR were not prepared, because, due to lack of symmetry, the transition dipole moments are most likely not orthogonally oriented inR⊥R, and therefore
Photophysical properties of compounds with parallel chromophores 13.2
300 400 500 600 700 800
0.0
Figure 48:Normalized absorption/emission spectra (solid/dotted curves) in CHCl3: a)B(105, blue curve) andR(112, red curve); b)B⊥B(107, black curve) and BkB(116, green curve); c) B⊥R(114, black curve) and BkR (118, green curve). The excitation wavelength was 348 nm for105,107and116, 530 nm for112and 360 nm for114and118. Emission spectra in (c) show an artefact at 720 nm due to higher order diffraction of the excitation light. This figure was published in an article with the title "Bichromophoric Compounds with Orthogonally and Parallelly Arranged Chromophores Separated by Rigid Spacers".[160]Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
As shown in figure 48, the spectra of compounds with blue fluorophoresB,B⊥B andBkB are almost identical. The absorption coefficients of bichromophores B⊥B andBkB are approximately twice as much as the absorption of B. The emission efficiencies of bichromophoric compounds are slightly reduced com-pared to the reference compoundB. The fluorescence lifetime is identical for all three compounds. A possible reason for the deviations could be aggregation of the bichromophores due to lower solubility. These results indicate that the two fluorophores inB⊥BandBkBcan be considered as isolated electronic systems without orbital overlap or the interactions can be neglected. Bichromophores B⊥RandBkRshowed exclusively fluorescence of the red fluorophore upon
ex-Chapter 13 BICHROMOPHORES WITH PARALLEL ARRANGED FLUOROPHORES citation of the blue fluorophore. Regardless of bichromophore geometry, highly efficient EET was observed.
13.3 DFT calculations for bichromophores B k B (116) and B k R (118)
Figure 49 shows the ground state geometry of bichromophore BkB after en-ergy optimization using density functional methods. For both chromophores the same conformation was chosen as for model compoundB(most stable one in the case ofB). The absorption and emission dipole moments were calculated using time-dependent density functional methods (see chapter 19 for details).
The absorption dipole moment of one chromophore (the chromophore pointing towards the−xdirection) was calculated directly for the ground state geome-try. The emission transition dipole of the other chromophore (the one pointing towards the+xdirection) was determined again by transferring it’s orientation from model compoundBto bichromophoreBkB. In fact, the transition dipole moments are parallel.
Figure 49:DFT optimized ground state geometry of bichromophore BkB(116). The absorption (S0→S1) and emission (S1→S0) transition dipole moments are shown as purple and blue double-headed arrows, respectively. H atoms are omitted for clarity.
The ground state optimized geometry of bichromophore BkR is shown in fig-ure 50. Also here, the same conformation was chosen for the "blue" chromophore as for model compound B (most stable in the case of B). The absorption and emission dipole moments were determined using time-dependent density
func-Estimation of energy transfer efficiency based on ideal Förster . . . 13.4
"red" chromophore was determined directly for the ground state geometry. The emission transition dipole of the "blue" chromophore was determined again by transferring it’s orientation from model compoundBto bichromophoreBkR.
Figure 50:DFT optimized ground state geometry of bichromophore BkR (118). The absorption (S0→S1) and emission (S1→S0) transition dipole moments are shown as red and blue double-headed arrows, respectively. H atoms are omitted for clarity.
13.4 Estimation of energy transfer efficiency based on ideal Förster point-dipole approximation for bichromophores B k B (116) and B k R (118)
The geometrical arrangement of the transition dipole moments was used to de-termine the orientation factorsκ2for bichromophoresBkBandBkR. Using the photophysical properties of model compoundsBandR, the energy transfer ef-ficiency based on the ideal Förster point-dipole approximation was evaluated.
The anglesθD, θA andθDA and distance |~RDA| between the emission and ab-sorption transition dipoles are given in table 12 (see section 1.2.1 for angle def-initions). Using equation 4, the orientation factorκ2was calculated (table 12).
Taking into account the spectral overlapJDAand the refractive index of CHCl3 nD(20°C)=1.4459,[134]the Förster radiiR0and the FRET efficienciesEFRETwere calculated (equations 5 and 6). The results are given in table 12.
Chapter 13 BICHROMOPHORES WITH PARALLEL ARRANGED FLUOROPHORES
Table 12:AnglesθD,θAandθDAbetween transition dipole moments and interconnect-ing vector~RDA, orientation factorκ2, distance between the donor and accep-tor|~R
DA|, Förster radiusR0, and estimated FRET efficiencyEFRET. Compound θD θA θDA κ2 |~RDA|/nm R0/nm EFRET BkB(116) 90.0° 90.0° 0.0° 1.00 1.33 2.93 0.99 BkR(118) 90.0° 83.2° 6.9° 0.99 1.33 3.61 1.00
These results validate the usefulness of DFT calculations. Indeed, in the case of parallel "blue" and "red" chromophores the expected value ofκ2is 1, and the cal-culated ones are 1.00 and 0.99 forBkRandBkB, respectively. As we have seen, in the case of "perpendicular" bichromophores, the calculations predict quite rea-sonableκ2values; In particular, forB⊥B, the predictedκ2-value is appreciably higher than that of bichromophoreB⊥R.
14 Time-resolved fluorescence
anisotropy experiments to study intramolecular EET
The intramolecular energy transfer in the new bichromophores was studied by (time-resolved) fluorescence anisotropy. These experiments and the data pro-cessing were kindly carried out by Dr. Mariano L. Bossi using a custom-made setup and custom-made software (see section 17 for details).
14.1 EET in bichromophore B ⊥ B (107)
To prove the presence and the power of intramolecular EET inB⊥Bbetween the identical chromophores in the case of nearly perpendicular chromopohore ar-rangement, time-resolved fluorescence anisotropy experiments were performed.
In order to control molecular rotation, the measurements were performed in bis(2-ethylhexyl)phthalate (DEHP) at temperatures between 4 °C and 60 °C. The high viscosity of DEHP at 4 °C (η≈0.28 Pa s)[161] almost excludes molecular ro-tation. The temporal evolution of the fl. anisotropy factor r(t) of compounds B⊥B,BkBandBis shown in figure 51.
Chapter 14 TIME-RESOLVED FLUORESCENCE ANISOTROPY EXPERIMENTS . . .
0.0 0.5 1.0 1.5 2.0 2.5
-0.2 -0.1 0.0 0.1 0.2 0.3 0.4
B
B B
B||B
r(t)
t / ns
Figure 51:Time-resolved fluorescence anisotropyr(t)of compounds B⊥B (107, red circles), BkB(116, blue circles) andB(105, black circles) in DEHP at low temperature (4 °C). This figure was published in an article with the title
"Bichromophoric Compounds with Orthogonally and Parallelly Arranged Chromophores Separated by Rigid Spacers".[160]Copyright Wiley-VCH Ver-lag GmbH & Co. KGaA. Reproduced with permission.
All three compounds (B,BkB, andB⊥B) show the same limiting anisotropyr0
att=0 (within the error of the measurement), which is close to the theoretical value 0.4 (emission of the photoselected fluorophores and excitation beam have same polarization). As expected forBandBkB, the anisotropy shows almost no decay. The very slow decay can be attributed to remaining rotations (as will be discussed later). In the case ofBkBintramolecular EET may occur, but does not affect the fluorescence anisotropy, because both fluorophores have the same ori-entation (θ=0° in equation 21, and thereforer1=r0in equation 22). In contrast, B⊥Bshows a fast anisotropy decay with a characteristic time of 163 ps (±50 ps), from an initial value of 0.32, and convergence towards a nonzero value (r∞≈0.1).
This is a strong indication for EET. The observed behavior can be explained by a fast energy hopping (back and forth) between identical chromophores. In this case the anisotropy evolutionr(t)can be described by equation 22.[101,102]
ForB⊥Bwithθ=90° andr0=0.36 a value ofr1≈–0.18 is expected. Directly after excitation, only emission of the initially excited chromophores (r0) is ob-served. Then, the energy transfer occurs in both directions, i.e. by energy
"hop-EET in bichromophoreB⊥B(107) 14.2 is well equilibrated (tk−t 1), an equilibrium anisotropy value r∞ is reached.
Equation 22 witht →∞gives
r∞ = r0+r1
2 . (30)
The observed values forr0≈0.32 andr∞≈0.1 are in fair agreement with the expected ones. Expected for the limiting anisotropy r0 is a value close the ob-served anisotropies forBandBkB, which are bothr0≈0.36. For the equilibrium anisotropy follows an expected value ofr∞≈0.09 (equation 22).
The average anisotropy calculated from the transients is also consistent with steady state anisotropy experiments (figure 52).
320 340 360 380 400
0.0
Figure 52:Excitation spectra (ITOT = Ik+2GI⊥; dashed lines) and measured steady state anisotropy (solid lines) of B⊥B (107) (red curves), BkB (116) (blue curves) andB(105) (black curves) in DEHP at 4 °C. The average anisotropy calculated from time-resolved anisotropy measurements is plotted as a dot-ted line for each chromophore. This figure was published in an article with the title "Bichromophoric Compounds with Orthogonally and Parallelly Ar-ranged Chromophores Separated by Rigid Spacers".[160] Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
The slow decay in the anisotropy (i.e.ττfl) of these three compounds (fig-ure 51) is due to rotations, which are not completely prevented in DEHP at 4 °C.
This was confirmed by measuring the anisotropy decay in DEHP at temperatures between 4 and 60 °C (see figure 58).
Chapter 14 TIME-RESOLVED FLUORESCENCE ANISOTROPY EXPERIMENTS . . .