High-performance liquid chromatography (HPLC)

Analytical and preparative HPLC were performed on a Knauer HPLC system:

Smartline pump 1000 (2×), UV detector 2500, column thermostat 4000, mixing chamber, injection valve with 20 µl loop for the analytical column and 200 µl loop for the preparative column; 6-port-3 channel switching valve; analytical column:

Eurospher 100 C18, 5 µm, 250×4 mm; preparative column: Eurospher-100 C18, 5 µm, 8×250 mm, 200 µl loop; flow rate: 1.2 ml/min (or 5 ml/min in the prepar-ative mode); solvent A: water + 0.1 % v/v TFA, solvent B: MeCN + 0.1 % v/v TFA;

gradient A/B: 70:30 to 0:100 in 25 min (unless stated otherwise).

17 Time-resolved fluorescence anisotropy measurements

PicoHarp (TCSPC Unit)

Sync Out

Ch0 Ch1

Computer

Laser Controller

APD P

375 nm

Fiber

L

P

L

L

S

L

F

Fiber

Scheme 30:Setup for time-dependent fluorescenece anisotropy measurements.

APD: avalanche photodiode; F: fluorescence band pass filter; L_1,4: focusing lenses; L₂: collimating lens, L₃: collecting lens; P₁: excitation polarizer;

P₂: emission polarizer; S: sample solution in a cuvette, placed inside a thermostat.

TCSPC measurements were performed in a custom-made setup consisting of a LDH-P-C-375 (blue chromophores excitation) or a LDH-P-C-510 (red chro-mophores excitation) picosecond pulsed laser (PicoQuant GmbH, Berlin, Ger-many), running on its internal clock at a frequency of 40 MHz, an ID100-50 (ID Quantique SA, Geneva, Switzerland) SPAD detector, and a PicoHarp 300 stand-alone TCSPC module. The sync signal used to compute the photon arrival times was provided by the laser driver. Count rates on the detector were adjusted to values below 10⁴Hz in all cases (typically ca. 10³Hz). An "L-shape" geometry was used with two Glan-Thompson polarizers (B. Halle Nachfl. GmbH, Berlin, Ger-many), a 440/40 nm or a 620/60 nm bandpass filter (AHF Analysentechnik, Tübin-gen, Germany) was placed in front of the detector, and diverse lenses to collimate the excitation light and to collect and focus the emission onto the detector were used. The temperature of the sample was controlled with a single Peltier cell holder (Varian Inc., Australia). The internal response function (IRF) was collected

Chapter 17 TIME-RESOLVED FLUORESCENCE ANISOTROPY MEASUREMENTS

the bandpass filter which was removed. Fluorescence decays were deconvoluted and fitted using custom made routines in MATLAB software.

18 Antibunching experiments

Scheme 31:Confocal microscope combined with two independent detection channels.

BS: beam splitter; D_1,2: avalanche photodiodes; DM: dichroic mirrors; F: flu-orescence band pass filter; HW: half-wave plate; L: lens; M: Mirror; OB: ob-jective; PH: pinhole; QW: quarter-wave plate. This scheme 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 per-mission.

A 375 nm diode laser (LDH-P-C-375; PicoQuant GmbH, Berlin, Germany) was fed to an optical fiber (PM-S405-XP; Thorlabs GmbH, Dachau/Munich, Ger-many) to generate a Gaussian beam. Circular polarization was generated, after the excitation light passed a pair of half- and quarter-wave plates. Excitation light was then reflected by a hot^†mirror (M254H00; Thorlabs) into an objective (HCX-PL-APO 100x/1.4-0.7 OIL CS; Leica Microsystems, Wetzlar, Germany) and the fluorescence was collected by the same objective and transmitted through the hot mirror. After a tube lens (f =200 mm), the fluorescence went through a 50 µm pinhole (correspond to ca. 1 airy disk diameter) and was collimated by another lens (f =100 mm), before it was split into two identical detection channels. Detection filters of type 460/60 (F39-46; AHF Analysentechnik AG, Tübingen, Germany) were used with single photon counting modules (COUNT

@blue; Laser Components GmbH, Olching, Germany) as detectors. The excita-tion repetiexcita-tion rate was 40 MHz and the laser power was ca. 21 µW. The detected signal was transferred through a time-correlated single photon counting card (DPC 230; Becker & Hickl GmbH, Berlin, Germany) and stored by a PC for post

†

Chapter 18 ANTIBUNCHING EXPERIMENTS

processing. Experimental control, data acquisition and analysis were performed with custom made MATLAB software.

19 DFT and TD-DFT calculations

To estimate the EET efficiency based on the Förster point-dipole approxima-tion (see secapproxima-tion 1.2.1), the orientaapproxima-tion of the absorpapproxima-tion and emission transiapproxima-tion dipole moments were calculated using density functional methods. All calcula-tions were carried out using B3YLP method^[165]with 6-31G(d,p) basis set in gas phase withGaussian 09^[166] software.

The method may be summarized as follows: First of all, the ground state geom-etry was optimized. To determine the absorption transition dipole moment, the electronic transitions were calculated for the ground state geometry using TD-DFT.^[167] For determination of the emission transition dipole moment, the ge-ometry of the S₁ state was optimized first, since that geometry is present when the photon is emitted (vertical transition). Then the electronic transitions (and transition dipole moments) between the ground and excited states were calcu-lated for the geometry of the excited state. The resulting emission transition dipole moment was obtained for the geometry of the excited state. To transfer it to the ground state geometry the adamantane structure was used as a refer-ence system. The absorption and emission transition dipole moments obtained in this way were placed at the geometrical centers of the fluorophores’ aromatic cores allowing to determine the distance between the transition dipoles and the orientation factorκ².

19.1 Ground state geometry optimization

For all molecules studied using DFT methods, the ground state geometry was op-timized first. The ground state geometry of the model compounds was opop-timized for all relevant conformers. The conformer with lowest energy was chosen for all further calculations. The bichromophore conformers were chosen to match the conformation of the corresponding model compounds.

The following code fragment of an input file root section was used typically for ground state geometry optimizations (_Opt keyword) followed by a frequency analysis (_Freqkeyword):

Chapter 19 DFT AND TD-DFT CALCULATIONS

1 %mem=32GB

2 %NProcShared=32 3 %Chk=jobname.chk

4 #n B3LYP/6-31G(d,p) Opt Freq 5

6 job description and comments 7

8 0 1

In some cases the optimization did not converge. In these cases the number of self-consistent field optimization cycles was increased using the keyword_scf=(

maxcycle=2000). The frequency analysis was performed to verify that there are no significant imaginary frequencies, i.e. to check that the optimized geometry represents a minimum of the potential energy surface. In case of imaginary fre-quencies, the optimization was repeated with stricter convergence criteria using option_Opt=tightorOpt=verytight.