Real-Time Monitoring of Photoinduced pH Jumps by In Situ Rapid-Scan EPR Spectroscopy

Abstract

This work represents the first demonstration of monitoring kinetics upon a light-induced pH jump by in situ rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy on the millisecond time scale. Here, we focus on the protonation state of an imidazolidine type radical as a pH sensor under visible light irradiation of a merocyanine photoacid in bulk solution. The results highlight the utility of photoacids in combination with pH-sensitive spin probes as an effective tool for the real-time investigation of biochemical mechanisms regulated by changes in the pH value.

Figure 1

(a) pH-sensitive nitroxide before and after cleavage of the photoprotecting group. (b) Photoacid. (c) Titration experiment: CW EPR spectra were taken at different pH values. Top to bottom: pH 1.0, 2.1, 3.1, 4.1, 5.1, 6.1, 7.2. (d) pH dependence of the isotropic hyperfine splitting measured as the distance between the low- and central-field components (circles). Fit to the Henderson–Hasselbalch titration curve (solid line).

Figure 2

Time-resolved rapid-scan experiment. (a) pH jump and corresponding decrease in the hyperfine splitting of the pH-sensitive nitroxide radical. Signal intensities are color-coded (view from above). A 100 G sinusoidal scan modulated at 10 kHz is used to acquire spectra with (a) 1 s or (b) 100 ms time resolution. The laser was switched on after 10 s of measuring time (450 nm, 3 mJ). (c) Slices along the magnetic field axis. Bottom to top: 1, 12, 12.1, 13, 45 s. (d) pH readout from the spectra in (a) circles and from the data shown in (b) dots.

Figure 3

(a) pH jump and thermal recovery. Laser irradiation (450 nm, 3 mJ) is indicated by a transparent horizontal. (b) Constant irradiation. (c) Influence of the sample composition and laser emission: 450 nm, 3 mJ, c(MCH) = 1500 μM (circles); 450 nm, 1 mJ, c(MCH) = 1500 μM (diamonds); 450 nm, 3 mJ, c(MCH) = 1125 μM (plus signs); 535 nm, 1 mJ, c(MCH) = 1500 μM (squares). All samples were irradiated after 10 s of measuring time.

Figure 4

(a) Change of the isotropic hyperfine splitting depending on the photoacid concentration: 1500 μM (circles), 1125 μM (plus signs), 750 μM (diamonds). Samples were irradiated at 450 nm and a pulse energy of 3 mJ. (b) Change of the splitting depending on the wavelength of the laser: 450 nm (circles), 525 nm (plus signs), 535 nm (diamonds), 550 nm (squares). The pulse energy was kept at 1 mJ.