It does not matter whether one is studying biological or chemical species, the flexibility and modular construction of the LP920 helps customize and integrate sophisticated upgrades and solutions in line with one’s requirements.
Using the LP920 users can precisely and consistently determine transient absorption either in kinetic and/or spectral mode and has a large sample chamber that will house a range of sample holders.
Transient Absorption and Photobleaching
The transient species presence may cause the sample to have either high or low absorption levels with respect to the ground state species absorption which is positive ΔOD and negative ΔOD respectively. While high absorption is linked with triplet-triplet or singlet-singlet transitions, a reduction in the measured optical density is linked with either ground state depletion or sample emission. The effects can normally be separated spectrally or based on their lifetimes. In certain cases like ruthenium, separation based on lifetimes is not possible.
Figure 1. Sample: Ruthenium bipyridine in water
Measurement Conditions: λpump=355nm, Epump=10mJ, pulsed probe source, λprobe=365nm (top picture), λprobe=450nm (bottom picture), 1 shot
Top picture: ground state depletion at 450nm
Bottom picture: transient absorption at 365nm
When too many excited states are generated, annihilation of excited states can take place due to high sample concentration or excessive pump energies whose lifetimes are long compared to the diffusion times of the molecules. Here, diffusion controlled collisions become possible resulting in the de- activation of both molecules. This example clearly shows the effect of laser energy on the transient dynamics. Annihilation is a non-exponential process but can be fitted with a series of exponential with the long lifetime showing the “true” excited state lifetime for the generated species.
Figure 2. Sample: Anthracene in cyclohexane (10-4M), partially degassed
Measurement Conditions: λpump=355nm, 3 different laser excitation pulse energies: Epump=50mJ (red), Epump= 10mJ (blue), Epump=1mJ (green).
Top picture: measured optical density data
Bottom picture: same data but scaled to the same peak height. The green curve represents a single exponential decay with a lifetime of τ = 118μs
Time Gated Spectra in Nanosecond Time Scale
Pyrene is characterised by distinct spectral bands when viewed time is resolved in the nanosecond time scale. In a time span of 50ns after laser excitation spectral features of fluorescence, singlet-singlet absorption and triplet-triplet absorption become evident.
Green curve: 10 ns after laser pulse. The spectrum shows the features of the ground state depletion (below 350 nm), fluorescence (370 – 420 nm) and the beginning of transient absorption bands (400 – 550 nm).
Red curve: 30 ns after laser pulse. At this time the fluorescence feature (370 – 420 nm) is almost diminished, ground state depletion is slightly decreased, transient absorption at about 415 nm is still rinsing, whereas transient absorption above 450 nm is beginning to decrease. Blue curve: 50 ns after laser pulse. Ground state depletion is continuing to decrease, triplet-triplet absorption at 415 nm is dominating, and the broad singlet-singlet absorption band continues to decrease.
Figure 3. Sample: Pyrene in methanol (10-4M)
Measurement Conditions: lpump=341nm, Epump=5mJ, pulsed probe source, 10ns gate width, 5 shots average per curve.
Time Gated Spectra in Microsecond Time Scale
The photocycle of the photoactive yellow protein shows kinetics throughout the nanosecond to millisecond time scale. In the microsecond time range, shown here, a transition from a red shifted to a blue shifted intermediate takes place.
Figure 4. Sample: Photoactive Yellow Protein
Measurement Conditions: lpump=450nm, Epump=10mJ, pulsed probe source, 50μs gate width, 5 shots average per curve, gated spectra from 50μs (violet curve) to 500μs (blue curve) after laser excitation in steps of 50μs
Time Gated Spectra in Millisecond Time Scale
At the end of the photocycle of the photoactive yellow protein both transient absorption features and ground state depletion are decaying to zero, enabling the protein to undergo a new photocycle. This is shown with time gated spectra in the millisecond time scale.
Figure 5. Sample: Photoactive Yellow Protein
Measurement Conditions: lpump=450nm, Epump=10mJ, contineous probe source, 50ms gate width, 5 shots average per curve, gated spectra from 50ms (brown curve) to 450ms (green curve) after laser excitation in steps of 50ms
Time Resolved Absorption Spectra
The computer controlled operation of the LP920-K Laser Flash Photolysis Spectrometer enables the user to generate Time Resolved Absorption Spectra in a two-fold process which is detailed below:
- Firstly, a series of transient absorption measurements over a pre-defined range of probe wavelengths is recorded
- Secondly, this data is sliced at desired time windows and delays from the laser pulse excitation
Automatic scanning through the spectral range causes a change in the probe background level. The changing background level does not have an effect on the value of the optical density, but it has an impact on the noise of the individual measurements. The LP920-K has the software option to either automatically reset the probe background offset or to fine-tune for this changing background level.
Figure 6. Sample: Anthracene in cyclohexane (10-4M), partially degassed
Measurement Conditions: λpump=355nm, Epump=20mJ, pulsed probe source, automatic scanning from 380nm to 480nm in steps of 1nm, spectral resolution 0.5nm, 10 shots per decay profile
Top: raw data obtained in kinetic mode, right: data after spectral slicing
Kinetic and spectral data sets can be viewed and manipulated in various ways using the L900 spectrometer software. 2D and 3D graphics are available as well as contour plot options. Data slicing converts a set of spectral data into a set of kinetic data and vice versa.
Figure 7. Sample: Ruthenium bipyridine in water
Measurement Conditions: λpump=355nm, Epump=10mJ, pulsed probe source, automatic scanning from 340nm to 670nm in steps of 1nm, spectral resolution 0.5nm, 5 shots per decay profile.
Top: data after spectral slicing, right: contour plot of the same data with the cross hair extracting more detailed spectral and kinetic information
Spectrally Dependent Transient Kinetics
Time resolved transient absorption spectra offers more information than kinetic measurements alone. The measurement of benzophenone in benzene shows two distinct absorption bands with the main band centre wavelength at 540nm and a second band above 600nm. The spectra for three different time intervals reveal that the long wavelength band shifts towards the near infrared spectral range with time. Blue curve: 0 -200ns, red curve: 600 – 800ns, green curve: 1.2μs – 1.4μs after laser excitation.
Figure 8. Sample: Benzophenone in benzene (10-4M) Measurement Conditions: λpump=355nm, Epump=40mJ, single shot measurements, step increment 1nm. Fit Results: at 540nm: τ = 340ns; at 660nm: τ1= 340ns (φ1 = 5%); τ2 = 4.8μs (φ2 = 95%)
Oxygen Quenching Of Transient Absorption Decays
The triplet states of organic molecules are often quenched by oxygen present in the solvent. Transient absorption measurements clearly reveal the sensitivity towards oxygen. The measurement example below shows the effect of different oxygen concentrations on the transient decay times, from 0% (blue curve) to 20% oxygen (black curve).
Figure 9. Sample: Erythrosin B in water Measurement Conditions: λpump=532nm, Epump=10mJ, pulsed probe source, λ probe=580nm, 10 shots average
About Edinburgh Photonics
Edinburgh Photonics is a manufacturer and supplier of an extensive range of instrumentation for Laboratory and Industrial use. These include:
- Spectrometers / Spectrofluorometers
(Steady State, Lifetime, Fluorescence)
- CO / CO2 Gas Lasers
- Pulsed / Optically Pumped Lasers
- Picosecond Pulsed Lasers and LEDs
- Additional Light sources
This information has been sourced, reviewed and adapted from materials provided by Edinburgh Instruments.
For more information on this source, please visit Edinburgh Instruments.