Chlorophyll for Efficient Solar Energy Capture Flashcards
What are the four phases of photosynthesis light reactions?
1) antennas and light collection
2) photochemistry (electronic energy converted to chemical energy via photons)
3) electron transfers stabilise chemical energy
4) storage of energy in new chemical compounds
Where is chlorophyll found?
-in high concentration in proteins in photosynthetic membranes which are folded to increase surface area
Atomic Bohr Model / Quantum Theory
- single electron states at different energy levels
- only photons with exact energy of transition (e.g. E2-E1) can be absorbed or emitted
Molecular Bohr Model / Quantum Theory
- electrons can interact with multiple nuclei which can rotate and vibrate
- a single electron state is split into many sub-states so transitions are possible for a range of energies
- the distribution of electrons across sub-states is governed by thermal equilibrium
Cholorophyll Electron Transitions
Absorption
- at room temperature, most electrons are near the bottom of the ground state
- most likely terminus after absorption is the centre of the excited state since the density of subtrates is greatest there
- this leads to broadening of peaks in the chlorophyll specta
- absorption is ultra-fast (fs)
Cholorophyll Electron Transitions
Fluoresence
- higher vibrational states are unstable and decay to the lowest vibrational state in picoseconds (energy loss as heat)
- this means that the most likely starting point for emission is the bottom of the exctied state
- the higher electronic state in the lowest vibrational state is relatively stable, decays in ns
- the most likely teminus for emission is the centre of the ground state
Cholorophyll Electron Transitions
Stokes Shift
- since ΔEa > ΔEf, the peak of absorption is at higher frequency than the emission peak
- the differenece in the location of the peaks is called Stokes Shift
Singlet Ground State, S0
- both electrons in ground state
- one spin up, one spin down
- spins are paired, anti-parallel
Singlet Excited State, S1
- one electron in ground state
- one electron in excited state
- one spin up, one spin down
- spins are paired, anti-parallel
Triplet State, T1
- with low probability (very slow) the S1 excited state can become a triplet state, T1
- one electron in ground state, one electron in excited state
- electron in excited state flips spins so that has the same spin as the ground state electron
- this is allowed under the Pauli exclusion principle since the electrons have different energies
- spins unpaired, parallel
Photoluminesence
Definition
- emission of a photon
- occurs by two mechanisms:
- -fluorescence
- -phosphoresence
Jablonski Diagram
Fluorescence
- photon emission from singlet excited state
- radiative
- timescale: 10^(-10) - 10^(-7)s
Jablonski Diagram
Phosphoresence
- photon emission form triplet excited state
- radiative
- timescale: 10^(-6)-10s
Photosynthesis and Triplet States
- triplet states can be highly reactive
- this can be damaging to biological systems since they’re so long lived
- they must be dealt with (or prevented) during photosynthesis
Absorption Spectroscopy
Experimental Set-up
- light source directed towards monochromator
- monochromatic light emerges at incident intensity, Io
- incident light passes through sample substance
- dectector measures intensity of transmitted light, I
Absorption Spectroscopy
Absorbance Equation
A = log_10_(Io/I) = εCx
-where A is absorbance, C is the concentration of sample molcules, x is the thickness of the sample and ε is the molar absorption coefficient
Absorption Spectroscopy
Estimating Absorbing Materials Concentration
-since A∝C, A can provide a simple estimate for concentration
Absorption Spectroscopy
Calculating the Molar Absorption Coefficient
- generally, ε increases with the number of pi binds
- for known concentration range, can plot A against C
- will be a linear relationship, A(λ) ∝ ε(λ) for constant concentration
- calculate ε form gradient
Absorption Spectroscopy
Absorption Spectrum
- plot of A against wavelength has important characteristics:
- -number, position and shape of peaks
- -wavelength of maximal absorption
- -width of absorption peaks
- the strength of a materials’ absorbancy is quanitified as a function of wavelength (or frequency)
Estimating Effective Absorption Strength
-integrate ε(λ) over the wavelengths of absorption
Chlorophylls Absorption
Wavelengths
- absorption over relatively large wavelength range, 400-900nm, requires extensive pi-system of conjugated bonds meaning a fairly large molecule
- when energy is absorbed it is distributed over the pi-system
- X and Y transitions for S0->S1 & S0->S2 means up to 4 absorption bands for one molecule
Chlorophylls Absorption
Energy Transfer Between Chlorophylls
- the lowest excited singlet state is sufficiently long-lived (τ~5ns) to allow energy transfer to neighbouring chlorophyll molecule
- theory and experiment show this τ allows energy propagation between assemblies of many chlorophylls over relatively large distances (10-100nm)
How stable is the excited state of a particular fluorophore?
-to answer this consider how long an electron stays in the excited state before decaying
-the lifetime of the excited state is equal to the reciprocal of the combined decay rates, typically 10ps-10ns
τ = 1 / (kf + kπr)
-where kf is the rate of radiative decay and kπr is the rate of all non-radiative dissipation
How bright is a particular fluorophore?
-consider reemission of photons vs. non-radiative dissipation
-fluorescence quantum yield is the rate of fluorescence over all other processes
-can be close to unity or <5%
φf = kf / (kf + kπr)
-where kf is the rate of radiative decay and kπr is the rate of all non-radiative dissipation
What does amount of fluoresence depend on?
- fluoresence depends on:
- -absorption i.e. concentration, ε
- -efficiency (or rate) of the competing decay processes
Intensity of Fluoresence
If = φf * Ia = kf / (kf + kπr) * Ia
-where Ia is the intensity of absorbed light, φf is the fluoresence quantum yield, kf is the rate of radiative decay and kπr is the rate of all non-radiative dissipation
Relative Fluoresence Intensity
If ~ Fr(λ) ∝ φf(λ)
If ~ Fr(λ) ∝ A(λ)
-most fluoresence spectrometers measure ‘relative’ fluoresence, Fr(λ), because it can be challenging to detect every photon in/out
Steady State Fluoresence Spectroscopy
- fluoresence measured at 90’ to light source so incident light isn’t picked up
- typically use a collection time of 10-60sec for reasonable signal to noise ratio
- fluoresence can be measured as a function of excitation wavelength with fixed emission position or function of emission wavelength with fixed excitation position
- if we excite a particular wavelength (e.g. where absorption is known to be high) then record fluoresence intensity at a range of emission wavelengths (integrate the signal over a defined time)
Measuring Fluorescence Quantum Yield
-with a special accessory added to the fluoresence spectrometer, absolute fluoresence quantum yield can be measured
-this is done by collecting every excitation vs emitted photon:
φf = kf / (kf + kπr) = no. of photons emitted / no. of photons absorbed
Time-Resolved Fluoresence Spectroscopy
- with an alternative instrument configuration we can measure fluoresence intensity as a function of time
- to do this we need to measure at a ps-ns timescale
- the measurement system needs a fast and well-defined light source which excites the sample coherently so the chromophore enters the excited state at a defined t=0
- use a pulsed laser with 50-100ps duration pulse
- uses timing electronics with a cumulative histogram graphical display
- need a single-photon-counting detector with high detection quantum efficiency which can be quickly rest ready for another photon, dead time
Fluoresence Decay Histogram
- using time-resolved spectroscopy repeat the time measurement many times and count how many photons have arrived after what time
- sort the photons into a histogram accordint to their arrival times
- exponential fit of fluoresence decay to obtain fluorescence lifetime i.e. number of molecules in the excited state decreases at a rate proportional to the current number of electrons in the excited state
Fluorescence Decay Curve
Equation
N(t) = No * exp(-kt) -where k is the rate constant -OR N(t) = No * exp(-t/τ) -where τ=1/k is the mean lifetime, the mean time at which the population of the assembly is reduced to 1/e~37% of the initial value
Fluorescence Decay Curve
Multi-exponential Fitting
F(t) = Σ Ai * exp(-t/τi)
-sum over i=1 to i=n
-e.g. a bi-exponential fit, if there are two decay processes
F(t) = A1exp(-t/τ1) + A2exp(-t/τ2) + z
-where τ1 is the lifetime of component 1, A1 is the amplitude of component 1 and equivalently for τ2 and A2
-and z is the fitting constant (detector background noise?)
Fluorescence Excited State Lifetime and Natural Excited State Lifetime
-the natural excited state lifetime is the excited state lifetime in the absence of non-radiative dissipation (knr=0), not possible to measure directly
τ0 = 1/kf
-the fluoresence lifetime of the excited state is the resiprocal of the combined decay rate of the excited state:
τf = 1 / (kf + knr) = τo*φf
What does relative fluorescence intensity depend on?
- emission wavelength obsrved
- excitation wavelength
- samples fluorescence QY
- sample’s concentration
- the detector’s quantum efficiency
- age of excitation lamp
Why is fluorescence relavant to studying photosynthesis?
- it helps us to understand the energy capture processes going on as part of ‘light harvesting’:
- -energy absorbed / energy of excited state
- -effectiveness of process (non-radiative decay ~wastage)
- -to quanitfy energy transfer efficiency, indirectly from fluoresence
Jablonski Diagram
Intersystem Crossing
- transition between vibrational levels belonging to electronic states of differnent spin multiplicity, e.g. S1 to T1
- non-radiative
- timescale: 10^(-10) - 10^(-6)s
Jablonski Diagram
Internal Conversion
- an electron in a higher energy singlet state transfers to a lower lying singlet state, it is immediately followed vibrational relaxation to the lowest vibrational level of the new electronic state
- non-radiative
- timescale: 10^(-11) - 10^(-9)s
Jablonski Diagram
Absoprtion
- transition from lower to higher electronic state, energy of photon converted to internal energy
- radiative
- timescale: 10^(-15)s
Jablonski Diagram
Vibrational Relaxation
- transition to lower vibrational level within the same electronic state
- non-radiative
- timescale: 10^(-12) - 10^(-10)s
Jablonski Diagram
Fluorescence
- transition from the lowest vibrational level of one electronic state to a lower electronic state of the same spin multiplicity
- radiative
- timescale: 10^(-10) - 10^(-7)s
