|Bielefeld University||Department of Chemistry||Biophysical Chemistry and Photochemistry||deutsch|
Time-Resolved IR and UV/Vis Spectroscopy
Absorption of light by a chromophore initiates a sequence of chemical reactions. Time-resolved spectroscopy is an analytical technique which allows us to elucidate these reactions by identifying intermediates and products and their kinetics.
In photosensors, the chromophore reacts with amino acids in the protein environment. These amino acids as well as changes in secondary and tertiary structure can be identified by applying spectroscopy.
We make use of the fact that the reaction of a chromophore changes characteristically its absorption in the visible range (its color) as well as in the IR range (its normal modes).
Fourier transform infrared (FTIR) spectroscopy allows us to investigate the structure and composition of chromophores, proteins, cells and a manifold of materials (see Angew. Chem. Int. Ed. 2010).
Using light-induced difference spectroscopy, we resolve reactions of chromophores and chemical processes of single amino acids against the background of thousands of normal modes of the complex (protein) environment and of water.
Advantages compared to UV/Vis spectroscopy are the smaller line width and the large number of transitions resulting in a much higher specificity and a clear identification.
Time-Resolved UV/Vis Spectroscopy
In time-resolved UV/Vis spectroscopy we record changes in absorption of white light by the chromophore after excitation with a laser pulse with a duration of a few nanoseconds.
The whole UV/Vis spectrum of the sample is recorded as a difference spectrum at different points in time from 60 nanoseconds to seconds after excitation and analyzed (see Photochem. Photobiol. 2011).
Time-Resolved FTIR Spectroscopy
We apply rapid-scan and step-scan techniques to elucidatee the mechanism of cyclic processes over a broad time range from 500 nanoseconds to seconds after excitation with a pulsed laser (see Biophys. J. 2009).
The step-scan method covers an important time range, which is not at all or only in principal accessible by ultrafast pump-probe spectroscopy.
The recording in stepscan is performed continuously with respect to both frequency and time and thereby leads to a gapless data set.
Data are analyzed by singular value decomposition and global fit, which yields a kinetic model of the reaction.
Method DevelopmentOne of our goals is to enable investigation of irreversible reactions and systems with long cycling times. Among other approaches, this goal is achieved by integrating microfluidic flow cells (see PCCP 2013).
We have demonstrated that the time resolution of DC step-scan can be enhanced by suitable electronic synchronization (see JACS 2015).
The employment of quantum cascade lasers allows us to enter totally new fields of application.
We analyze reactions of monolayers with nanometer thickness by ATR spectroscopy.
We compensate for the lacking spatial information of IR spectroscopy on proteins by double difference spectroscopy:
- segment-resolved double difference spectroscopy can assign signals to specific secondary structural elements or domains in proteins (see Biochemistry 2010 Nucleic Acids Res. 2016).
- by isotope labeling and double difference spectroscopy we address single amino acids in a collaboration with theory (see Sci. Rep. 2016).
For interpretation and assignment of the infrared spectra, we also perform quantum chemical calculations including explicit models for the environment. Even difference spectra can be calculated (see J. Phys. Chem. Lett. 2010, PCCP 2013).
For very demanding questions and the calculation of double difference spectra we are relying on collaborations (siehe Sci. Rep. 2016).