A protocol for determining cage-escape yields using nanosecond transient absorption spectroscopy

Summary Here, we present a protocol for the determination of cage-escape yields following excited-state electron transfer between a photosensitizer and a quencher. We describe steps for determining changes in molar absorption coefficient of the different oxidation states via photolysis experiments and the percentage of reacted species via steady-state or time-resolved spectroscopy. We then detail measurement of the amount of formed product via nanosecond transient absorption spectroscopy. For complete details on the use and execution of this protocol, please refer to Ripak et al. (2023).1


Ludovic
Troian-Gautier ludovic.troian@uclouvain. be Highlights A protocol to determine cageescape yields in solution is described Protocol is compatible with oxidative or reductive excited-state electron transfer Two approaches used depending on the photosensitizer's and quencher's stability Photolysis as alternative to spectroelectrochemistry and chemical redox reactions SUMMARY Here, we present a protocol for the determination of cage-escape yields following excited-state electron transfer between a photosensitizer and a quencher. We describe steps for determining changes in molar absorption coefficient of the different oxidation states via photolysis experiments and the percentage of reacted species via steady-state or time-resolved spectroscopy. We then detail measurement of the amount of formed product via nanosecond transient absorption spectroscopy. For complete details on the use and execution of this protocol, please refer to Ripak et al. (2023). 1

BEFORE YOU BEGIN
The protocol describes the specific steps to determine cage-escape yields following excited-state electron transfer between [Ru(bpy) 3 ] 2+ (the photosensitizer, PS) and 4-methoxy-benzene diazonium (the quencher, Q), using [Ru(bpy) 3 ] 2+ as actinometer, i.e., a species used to gauge the radiation intensity. The experiments were performed in acetonitrile, but any other inert, non-absorptive solvent can be used as long as they allow a complete solubility of the photosensitizers and quenchers. Note that, although described for oxidative electron transfer, this method was also used to determined cage-escape yields of reactions proceeding via reductive excited-state electron transfer.  We describe the sequential steps that are needed to perform these cage-escape yields measurements. The first part consists in determining changes in molar absorption coefficient of the photosensitizer in different oxidation states using photolysis. The second step describes how to determine the percentage of quenched photoluminescence and how to measure the concentration of products formed after excited-state electron transfer. Two methods for the determination of cage-escape yields are reported. One method uses one cuvette of PS with increased concentrations of quencher while the other method uses individual cuvette for each concentration of quencher.

Photolysis experiment
Timing: 0.5-1 h Photolysis experiments are performed to determine Dε PS+ , i.e., the difference in molar absorption coefficient between the PS in its ground and oxidized states (ε PS+ -ε PS ). This value is required to determine cage-escape yields and will be further discussed in the quantification and statistical analysis section (vide infra). The light-induced reaction proceeds according to the following equations:  (20-60 mM) with an excess (at least 100 times more concentrated) of sacrificial quencher are recorded at selected time intervals following irradiation until absorption changes are no longer observed ( Figure 1). Conversion of absorption spectra into molar absorption coefficient via the Beer-Lambert Law allows us to calculate the corresponding Dε spectra and determine the value of Dε for any wavelength of interest within the absorption limit of the Beer-Lambert law. Similar experiments can be performed for reductive electron transfer using electron donors such as EDTA, triethylamine or BIH for example. 36 Note however that reversibility of the reaction must also be assessed and confirmed to obtain rigorous results (vide infra).  2. Place the solution in a (quartz) cuvette with four optical faces and seal it with a septum and parafilm. a. Purge the solution with argon for 10 min and record an absorption spectrum in the appropriate wavelength range. b. Open the cuvette, quickly add 22mg of 4-methoxy-benzene-diazonium (30 mM). c. Reseal the cuvette with a septum. d. Parafilm and purge the solution with argon for 2 min. e. Record another absorption spectrum before starting the photolysis. 3. Irradiate the cuvette with a LED strip or LED lamp at a wavelength close to the photosensitizer's l max in the visible region.
4. Record a UV-Vis absorption spectrum at selected time intervals until no further changes are observed. At this point, the absorption spectrum of the mono-oxidized PS is obtained.
Optional: After complete oxidation, remove the septum and add an excess of electronacceptor, L-ascorbic acid in this case, to assess the redox process reversibility. If the PS is stable in its oxidized form, and that the electron donor does not absorb it that range, the original spectrum should be recovered. If reductive electron transfer was investigated, an electron acceptor needs to be added, such as K 2 S 2 O 8 , for example.
Note: All the L-ascorbic acid might not be soluble in acetonitrile. It is important to let the solid settle before recording an absorption spectrum to avoid any scattering.
Note: The cuvettes used in the present study have a 24/40 joint that can be easily sealed with a septum, Teflon tape and parafilm.
Note: Purging is performed using an argon tank connected to a bubbler filled with acetonitrile and is then connected to the cuvette. This allows to control the gas flow as well as avoid changes in concertation due to evaporation. Nitrogen can also be used instead of argon following the same procedure and controls.
Note: Dε PS+ are more adequately determined by spectroelectrochemistry, 37-39 but this equipment is not always available in the laboratory. The photolysis approach presented here is an affordable and reliable alternative to determine Dε PS+ . Alternative methods using chemical oxidant are also possible. 39,40 Determining the optimal operating wavelengths Cage-escape experiments require multiple spectroscopic measurements at different wavelengths. l exc is the excitation wavelength that must be common for both the actinometer and the sample. l det is the wavelength at which the transient absorption signals will be detected. l det can vary for each photosensitizer and can be different from the actinometer. CRITICAL: l exc should be different from 450 nm that is used as the l det for the actinometer in the present case.
Note: In the example in Figure 2A, the highlighted green zone represents the wavelengths that can be used to excite [Ru(bpy) 3

MATERIALS AND EQUIPMENT
Nanosecond transient absorption measurements were recorded on a LP980-K spectrometer from Edinburgh Instruments equipped with an iCCD detector from Andor (DH320T). The excitation source was a tunable Nd:YAG Laser NT342 Series from EKSPLA. The third harmonic (355 nm) at 150 mJ was directed into an optical parametric oscillator (OPO) to enable wavelength tuning starting from 410 nm. The laser power was then attenuated to reach appreciable signal/noise and the integrity of the samples was verified by UV-Vis measurements. The LP980-K is equipped with a symmetrical Czerny-Turner monochromator. For single wavelength absorption changes, a 1800 g mm -1 grating, blazed at 500 nm is used, which affords wavelength coverage from 200 to 900 nm. For spectral mode (iCCD), a 150 g mm -1 grating, blazed at 500 nm is used, offering a wavelength coverage of 540 nm over the full wavelength range extending from 250 to 900 nm. Single wavelength absorption changes were monitored using a PMT LP detector (Hamamatsu R928) which covers the spectral range from 185 to 870 nm. The probe was a 150 W ozone-free xenon short arc lamp (OSRAM XBO 150W/CR OFR) that was pulsed at the same frequency as the laser. An average of 30 scans per measurement was used. Note: The quencher solution is prepared just before the beginning of the experiment. If the quencher is not used immediately after weighing, make sure to keep it under stable storing conditions (refrigerated (at 4 C) in dark conditions for this example).

Note:
The quencher solution is prepared in the PS stock solution to prevent changes in absorption due to dilution during the titration.

Actinometer calibration
Timing CRITICAL: Efficient Argon-purging of the analyzed solution is a key parameter to control errors on the experiments. Measuring the actinometer excited-state lifetime as a function of purging time until it remains unaltered will help to find the minimum time needed to efficiently purge the solution.

Calibrate the laser power by nanosecond transient absorption measurement.
Note: Calibration is performed by using different laser intensities in order to define the optimal laser power ( Figure 3). Here, a 60% laser intensity was used, corresponding to a laser power of 14 mJ/pulse.

OPEN ACCESS
Note: Saturation can appear with less than 100% laser power. DAbs signal should increase linearly with laser power intensity. If the intensity of the signal remains unaltered upon increasing laser power, saturation of the excited state has been reached and a less intense laser power should be used. This parameter depends on the laser itself. It is preferable to work with a moderate laser power to ensure the correct calibration of the actinometer.
6. Record the single-wavelength absorption changes of the actinometer using the previously determined laser power as well as l exc and l det .
Note: It is recommended to repeat step 6 at regular intervals during the experiment to ensure constant laser power irradiation.

Timing: 1 h per experiment
Two methods can be used to record cage-escape yields using a combination of steady-state or timeresolved photoluminescence and nanosecond transient absorption spectroscopy. The choice between the two methods depends on the relative stability of the photosensitizer and quencher in the dark and under irradiation. The first method consists in preparing one spectrophotometric cuvette with the photosensitizer and adding multiple aliquots of quencher solution. The second method consists in preparing a series of spectrophotometric cuvettes with an identical concentration of photosensitizer and different concentrations of quencher. Note: It is important to work in a concentration range that allows significant variations of the quenching percentage and DAbs with increasing quencher concentration.
Note: The timescale used to record the single wavelength transient absorption changes is increased compared to the one used for the actinometer. In this case, a timescale of 100 ms was used.
CRITICAL: The cuvette orientation was also controlled in the different devices. One face of the cuvette was marked and was always oriented in the same direction in the sample holder of the different devices.  i. Record the absorbance at l exc of the spectrophotometric cuvette with the quencher. j. Record a steady-state photoluminescence spectrum (a complementary excited-state lifetime decay can also be recorded if desired) of the spectrophotometric cuvette with the quencher (Figures 4B and 4D). k. Record the single wavelength (l det ) transient absorption changes ( Figure 4F). l. Repeat steps c-h with the other cuvettes but change the added volume of quencher stock solution (step h) to 200, 500, 1000 and 1500 mL respectively.
Note: It is important to work in a concentration range that allows significant variations of the quenching percentage and DAbs with increasing quencher concentration.
Note: The timescale used to record the single wavelength transient absorption changes is increased compared to the one used for the actinometer. In this case, a timescale of 100 ms was used.
CRITICAL: Sealing tightly the spectrophotometric cuvettes is of paramount importance for these measurements as oxygen can impact the amount of excited-state quenching, product formation and recombination processes after electron transfer. It is advisable to use Teflon tape, a new septum and parafilm to seal the cuvette for each measurement.
CRITICAL: The cuvette orientation was also controlled in the different devices. One face of the cuvette was marked and was always oriented in the same direction in the sample holder of the different devices.
Note: Method 1 is less time-consuming and more convenient when no stability issue arises (see troubleshooting). Method 2 is more consuming in terms of materials and time but allows to limit problems due to stability issues.

EXPECTED OUTCOMES
This protocol allows us to determine the cage-escape yields, i.e., the efficiency with which the formed geminate radicals pair separate and escape the solvent cage after excited-state electron transfer. With the values obtained herein, the operators should be able to determine cage-escape yields around 35% for the light-activated electron transfer reaction between [Ru(bpy) 3 ] 2+ and 4-methoxy-benzene diazonium tetrafluoroborate.

QUANTIFICATION AND STATISTICAL ANALYSIS
In here, we describe how the above-mentioned protocols and corresponding collected data are used to determine the cage-escape yields. Final cage-escape yields (4 CE ) values are obtained by comparing the relative yield of PS + produced (4) to the percentage of quenched photoluminescence (%PL) determined by steady-state ( Figure 5A) or time-resolved photoluminescence ( Figure 5B). A concatenated linear regression of all the data points by constraining the Y-intercept at 0 provides a slope that corresponds to the cage-escape yield. R 2 of 95% and 90% are obtained for the steady-state and time-resolved data, respectively. Cage-escape yields of 34% and 32% are obtained using the steady-state and time-resolved data, respectively.

LIMITATIONS
There are several limitations that may complicate such experimental protocol or render it unreliable. The first one is that the photosensitizer and the quencher must remain stable (i.e., no degradation or formation of side-products) in the dark and upon irradiation. Compounds that degrade would impact the absorbance as well as DAbs and hence impact the final cage-escape yields. The method that is proposed here to use individual spectrophotometric cuvettes with fresh concentration of  3 ] 2+ in the presence of 4-methoxy-benzene diazonium tetrafluoroborate. Experiments are presented for operators 1 and 2 (Op.) according to methods 1 and 2 (Met) and the indicated trial. The slope was used to extract a cage-escape yield of 34% (a) and 32% (b). An error of ca. 10% is estimated based on uncertainties associated with the changes in molar absorption coefficient of the actinometer and the oxidized photosensitizers as well as with the trial averages. An operator unfamiliar with these experiments also obtained 4 CE of 34% and 32% using method 1 and 2 of this protocol, respectively.

OPEN ACCESS
quencher and PS allows us to potentially remediate this limitation. An additional experimental limitation is that the photosensitizer and the actinometer must absorb at the same wavelength. This is usually the case when [Ru(bpy) 3 ] 2+ is used as actinometer but may represent a limitation in some cases. Finally, the changes in absorbance between the photosensitizer in the ground state and the photosensitizer in its oxidized or reduced state must be sufficiently large to accurately determine a Dε value and the corresponding single wavelength absorption changes.

TROUBLESHOOTING
Dark reactivity of the photosensitizer/quencher pair Because cage-escape experiments require relatively large concentration of both the photosensitizer and the quencher, a deterioration of the sample or the quencher solution may sometimes be observed (step 7.e). This was observed for some photosensitizers in the original report where a notable change in the absorption value at l exc was observed, indicative of ground-state oxidation. 1

Potential solution
The quencher can be dissolved in solvent alone (the same as for the PS solutions) but a dilution factor should be considered for the %PL Quenched values since the concentration in PS will vary with the additions. Method 2 (step 8) is preferred when using this option. If a side-reaction is occurring within the time frame of the experiment, then the results are unfortunately unreliable and should not be used.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact. Ludovic Troian-Gautier (Ludovic.Troian@uclouvain.be)

Materials availability
This study did not generate new unique reagents.

Data and code availability
The published article includes all datasets generated or analyzed during this study.

AUTHOR CONTRIBUTIONS
A.R. and S.D.K. conducted the experiments. L.T.-G. and B.E. conceptualized, designed the experiments, and supervised the research. All the authors analyzed and interpreted the data, wrote, and edited the manuscript.

DECLARATION OF INTERESTS
The authors declare no competing interests.