Post on 23-Mar-2020
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Femtosecond resolved absorption spectroscopy
P R E S E N T A T I O N B Y : R O S H N I B A N O
2 8 T H O C T O B E R , 2 0 1 1
General use of absorption spectroscopy
Determine absorption spectra of photoactive agents
Characterise excited states, reactive intermediates and photoproducts
Sequence of events, rates of occurrence and factors influencing rates
- Ground state absorption – for stable
(1ms) species, CW light
- Transient absorption – for metastable
- species (fs to ks), pulsed laser + probe
source (CW or laser)
Picture from : http://www.photobiology.info/Nonell_Viappiani.html
Time resolved absorption spectroscopy
Steady state spectroscopy – continuous irradiation, continuous creation of excited states and finally steady state
Time resolved spectroscopy – light source’s intensity fluctuates in time, burst of excited states (only a fraction of them excited), monitor time evolution
-- fourier transform limited, therefore time resolution sacrificed for spectral resolution
Femtosecond time resolution
Timescale of electronic transitions in most photochemical/photophysical processes
Large bandwidth for small pulses
Such sources are prepared using “mode locking”
Can be used to generate probe pulses by chirping
1mJ pulse with 100fs duration – 10 GW !
Picture from :(Lecture notes) Basics of femtosecond laser spectroscopy, Mikhailovsky, UCSB
The pump probe approach
Excitation with a pump laser beam (pulse duration shorter than time constant of reaction)
Sample interrogated with probe before and after excitation Can monitor absorbance change at given wavelength or at several
wavelengths Can delay probe pulse wrt pump pulse for studying time evolution Study disappearance of excited states, formation of reactive
intermediates and photoproducts
Picture from : Berera, PhotosynthResearch, 2009
Picture from : http://os.tnw.utwente.nl/images_new/proj40_2.jpg
Some applications of fs-resolved spectroscopy
Novel spectral features may lead to discovery of reaction intermediates
Figure out reaction mechanisms Rate constant of each kinetic step Temperature dependence, for
example, activation parameters
Picture from : Introduction to femtosecond laser spectroscopy and ultrafast XRD , Wolf, Freie Universitat Berlin
Protein found in membrane of archaean that lives in salt marshes
Aerobic – makes ATP through ETS, Anaerobic – makes bacteriorhodopsinbR and harvests light to set up a proton gradient
Light absorbing chromophore – retinal (polyene compound) linked that isomerises from all-trans to 13-cis form and starts a thermal cascade
In methanol solution, all-trans goes mostly to 11-cis with quantum efficiency of 0.15. In bR, goes to 13-cis with quantum efficiency of 0.6 Stored energy is used to pump protons across the membrane!
Highly specific, quick isomerisation – how?
The model before this paper came…
Photoexcitation triggers ultrafast movement towards excited state minimum (“twisted conformation”)
Twisted conformation can go to all-trans or 13-cis
Ultrafast torsional motion must be
along a repulsive potential in the Franck Condon region
● Contrasts with retinal in solution where FC region = minimum of excited state due to symmetry across C13-C14 bond
● Repulsive potential can result only if protein shifts ground and excited state landscapes wrt each other
But bR mutants have quantum efficiencies similar to bR, even with different excited state lifetimes!
Stimulated emission dynamics
If the FC region of excited potential energy surface is repulsive, can monitor time dependence of stimulated emission. Will redshift as photoexcited retinal progresses towards minimum of surface.
(LEFT) Stimulated emission and excited state absorbance decay, ground state bleach recovers, photoproduct grows. (RIGHT) No red shift in stimulated emission with time !
Excited state dynamics
Both stimulated emission and excited state absorbance come from the same state, therefore dynamics should be the same.
Modelled with biexponential –fast and slow decay components - 370 fs (87%) and 2.1 ps(13%) – indicates multiple species.
-- Since stimulated emission spectra are identical at different delay times, the kinetically different populations are spectroscopically similar.
Average rise time – 10 fs
The missing stimulated emission !
Integrated stimulated emission should be comparable to integrated ground state bleach, but isn’t.
Subtract ground state bleaching and stimulated emission to get only excited state absorption
The stimulated emission is masked by uncharacterisedpositive going absorbance, and hence seems to be lower!
The final three state model
Rise time of stimulated emission (10 fs) suggests flat, not repulsive FC region
Two avoided crossing regions (marked by red arrows)
S1 and S2 strongly overlap – affects crossing of second avoided region
Non exponential relaxation due to conformational heterogeneity of S0
which modulates the barrier leading to reactive surface of S1
(encircled)
In conclusion, protein does not play an important role in photo-isomerisation; just catalyses conversion along C13-C14 bond and inhibits that along others. It is the intrinsic electronic energy landscape of retinal which leads to high efficiency photoisomerisation.
Thank you!
Acknowledgements
Images in first slide from :- (Left) http://sherwingroup.itst.ucsb.edu/img/image006.gif
- (Right) Textbook of Physical Chemistry, P W Atkins
Useful readings :
- http://www.photobiology.info/Nonell_Viappiani.html
- (Lecture notes) Basics of femtosecond laser spectroscopy, Mikhailovsky, UCSB
- (Lecture notes) Introduction to femtosecond laser spectroscopy and ultrafast XRD , Wolf, Freie Universitat Berlin
- (Lecture notes) Time resolved absorption spectroscopy, Penzkofer
- Ultrafast transient absorption spectroscopy : principles and application to photosynthetic systems, Berera et. al., Photosynth Res (2009), 101 : 105-118
Cool Youtube video on femtosecond pump-probe spectroscopywww.youtube.com/watch?v=mdNr6eVBJqk