Chm2922 - Fluorescence

Fluorescence: How will emission/fluorescence intensity be affected if concentration of sample is doubled?

Emission/fluorescence intensity would double as well because intensity is linear with concentration (if abs < 0.1)

A rhodamine based compound fluoresces bright yellow when GREEN laser light is directed at it. (a) Why is there a difference between absorption and emission wavelengths? (b) What is this energy difference known as? Where does this energy go? (c) What colour do you think the fluorescence emission will be if blue laser was used instead? Why? (d) What do you think will happen if red laser is used?

(a) Following excitation (via absorption of photon), a molecule loses energy non-radiatively via vibrational relaxation (brings it to lowest vibrational state of first excited state) before fluorescence occurs. HENCE, absorption and emission would NOT be the same. (b) Stokes Shift. Due to loss of energy to non-radiative process, photon emits at a longer wavelength. Energy dissipates into the surroundings, usually lost as heat. (c) Fluorescence will STILL be yellow because once it gets excited, the molecule will experience non-radiative loss of energy via vibrational relaxation --> brings to lowest vib. state of the first excited state. Fluorescence will then occur at this level so it is always the same energy emitted (colour) (d) Red has a wavelength of 700nm while rhodamine absorbs at 250-350nm. The longer the wavelength, the less the energy. Hence, red has insufficient energy to excite photons in rhodamine.

What order of kinetics does fluorescence follows?

- 1st order kinetics: reaction depends on concentration of one reactant - Concentration of excited state molecules at time (t) = Initial concentration x exp^(-t/decay rate) ----> determines probability that a given excited state has decayed after a time has passed

What is fluorescence quenching for?

- An important technique for measuring BINDING AFFINITY between ligands and proteins. - Fluorescence quenching is the decrease in the quantum yield of fluorescence, induced by a variety of molecular interactions with quencher molecule(s).

Why are phosphorescence quantum yields usually very low at room temperature?

- At room temperature, non-radiative relaxation usually dominates and its just faster to fluoresces. - At low temperature, vibrational relaxation is MUCH SLOWER, so phosphorescence can compete and take place instead of fluorescence

fluorescence lifetime

- Average time fluorophore remains in excited state before returning to ground state and emitting a photon

Describe how quinine can be quenched by chloride ions

- Collision transfers energy from EXCITED quinine --> Cl- - THEN, quinine continues to lose it non-radiatively through the other internal processes

How to measure fluorescence spectra

- Commercial fluorimeter - Xe lamp for excitation - 90 degree sample arrangement - Monochromators (gratings) for wavelength selection + controllable slit widths - Highly sensitive photomultiplier detector

What can chemiluminescence be used for?

- Determination of Ozone cos it is sensitive to < 1 ppb O3 and can provide a LINEAR response up to 400ppb

What is the luminol system?

- Direct chemiluminescence mechanism - Luminol WITH strong oxidants (O2, H2O2) AND catalyst (Fe(II)/ Fe(III)/Co(II)/peroxidase) = chemiluminescence

Process after photon is absorbed

- Electron promoted from ground state (S0 to a higher molecular orbital - Spin is conserved (S=0) - S1 = 1st excited level - S2 = 2nd excited level

Intermolecular quenching, what is it? What is it good for?

- Excited states can be de-activated EXTERNALLY. By collision with another outside species. - For example: fluorescence from quinine can be quenched by chloride ions. This can be used to measure concentration of QUENCHER

Application of the luminol system

- Field screening for invisible blood stains - Spray area with HCl to decompose haemoglobin --> release Fe(III) - Fe(III) detected by adding strong oxidant (H2O2/O2) + luminol = chemiluminescence!!!!

What can the Stern-Volmer plot be used for?

- Gradient = Ksv - Can be used to read off conc. of quencher for an unknown solution - Can use either fluorescence intensity/lifetime - Can get rate constant for quenching if emission intensity WITHOUT quencher

Why are rhodamines ideal as reference compounds

- Has a quantum yield approaching 1.0/100% = displays one of the brightest emissions - Can be easily detected even at low concentrations SOO its known

What does it mean to have a quantum yield of 1.0 (100%)

- Higher the fluorescence quantum yield, the stronger the fluorescence signal intensity (it means that with each proton absorbed, results in a photon emitted)

Why is fluorescence useful?

- Highly sensitive at even low levels - Minimally invasive - Time-resolved spectroscopy: using fluorescence to find out what is happening (e.g. photosynthesis, dye sensitised solar cells) - Fluorescence experiments: can measure rate constants for energy and electron transfer

Triplet states and intersystem crossing (ISC)

- Intersystem crossing (ISC) is a competitive process that can de-activate S1 --> leading to triplet state, T1 - In triplet states, electrons have to be in different orbitals

What is kq and how do you calculate kq?

- Kq: Rate constant of additional quenching process - Kq = (1/Tflu) - (1/Tref) Tflu: observed fluorescence lifetime Tref: lifetime without quenching

Quantum yield by reference method

- Measure quantum yield of a sample against a reference standard of known quantum yield - Reference must absorb and emit over a similar excitation wavelength range and has a similar quantum yield (assumed to be absorbing same number of photons) - Reference can be used in a different solvent (convenient), but if refractive index is different then it will need to be corrected - Conditions must be identical - Low absorbance (<0.1)

Self-Quenching

- Occurs at high concentrations (A>0.1), this causes non-linear behaviour caused by COLLISIONS between EXCITED molecules = radiation-less energy loss

Why is phosphorescence slower than fluorescence?

- Phosphorescence is spin forbidden (whereas fluorescence is spin allowed), cannot have change of spin - Phosphorescence involves a molecule in an excited triplet state relaxing to a singlet ground state. (fluorescence is from excited singlet state to singlet ground state. triplet state is lower in energy so molecule would need to lose MORE energy first before it phosphoresces)

Define QYflu using rate constants for excited state process

- QYflu = kr/ (kr+ sumKnr) - Knr: sum of all the non-radiative ways for a molecule to relax (e.g. vibrational relaxation, intersystem crossing, internal conversion, etc)

Describe QY flu in terms of photons absorbed and emitted.

- QYflu = photons emitted/photons absorbed - The probability of an excited molecule relaxing by radiative emission

A 20micrometre solution in toluene registers 12.5k counts, but increasing its concentration results to an increase in intensity as well. However, as concentration was increased further, the intensity counts became lower. Why is that so? Suggest reasons.

- Relationship between fluorescence intensity and concentration is not linear - It is only linear at lower concentrations, at higher concentrations this relationship starts to deviate from linearity. - This is because of Beer Lambert's Law, in which fluorescence intensity is linear with fraction of light absorbed. But as concentration increases, intermolecular effects such as collisions and inner-filter effects (self-absorption because of overlap of emission wavelength with absorption oops) = reduce fluorescence intensity even further.

Characteristics of molecules with high quantum yields for fluorescence

- Rigidity and extended conjugated regions - Planar unsaturated rings

Factors that affect fluorescence

- Rigidity: Increases quantum yield by decreasing non-radiative loss (through vibrational relaxation). The opposite applies as well, if flexibility increases then quantum yield decreases - Temperature: Quantum yield decreases as temp increases --> more electronic collisions and deactivation - Heavy atom solvents: Solvents containing I or Br = decreases quantum yield because it enhances intersystem crossing (enhanced phosphorescence) - pH: Changes resonance stability by changing ionization

Advantages of Chemiluminescence detection

- Simple instrumentation (no filters, monochromators) - Highly sensitive BECAUSE there is NO background light emission (ppm-->ppb) - Linear signal: Conc relationship over several orders of magnitude

Advantages of chemiluminescence over fluorescence

- Simpler instrumentation - No background - Wide LINEAR range with concentration

phosphorescence

- Spontaneous emission of a photon from excited triplet state (T1), this state is lower than the 1st singlet state - BUT at room temperature, non radiative relaxation dominates over the phosphorescence

Non-radiative Relaxation

- loss of internal energy occurs in a series of small steps (vibrational relaxation) until it gets to the bottom of an excited electronic state - Internal Conversion (IC), occurs to get the system into the next lowest electronic state (S2 --> S1) - IC and Vibrational relaxation occurs faster than emission, however due to the large energy gap between S0 and S1, IC is much slower and fluorescence can take place instead.

What does the sum of the QY of all the processes that are available to deactivate a state add up to?

1

How to calculate emission intensity without quencher? (T0)

1/ (constant rate at which fluorescence occurs + sum of all constants of non radiation relaxation)

Quantum Yield of Fluorescence

= total photons emitted (I) / total photons absorbed (I0) - Probability of a photon out for each photon in - Area under emission spectrum proportional to photons emitted

What is luciferase

An enzyme, catalyses reaction in the presence of oxygen + luciferin Found in marine bacteria

What is the peroxyoxalate system?

Aryl oxalate esters oxidised hydrogen peroxide - Indirect chemiluminescent system (because fluorophore is present in ground state, in the example below it is the dye) + most efficient Example of this system: - Glow sticks. Dyes mixed in phenyl (aryl) oxalate. This mixture kept APART from hydrogen peroxide (usually in glass tube) until u break it = glow. - Reaction can be sped up by using heat.

Selectivity of luminol system

Chemiluminescence is activated by a variety of different catalyst, so it might not be THAT selective If there is the presence of Fe(II) and Cu(II), these can still catalyse the luminol reaction bah

Which is faster: fluorescence or (vibrational relaxation and internal conversion)?

Definitely NOT fluorescence

Stokes shift theory for fluorescence

Due to loss of energy to non-radiative processes, emission is always at a longer wavelength (lower energy) than excitation = fluorescence Difference between wavelength (energy) of the absorption maximum and emission maximum

Excitation spectra looks similar to absorption spectra. Why?

Excitation is equivalent to absorption since upon absorption, the molecule reaches the excited state Sn.

What are the processes that deactivate the excited state with their own rate constants?

Intersystem crossing (to triplet state), non-radiative relaxation, internal conversion and quenching processes - All the above are NON-radiative and SHORTENS the lifetime of the excited state and THUS measured fluorescence lifetime

Stern-volmer rate constant (Ksv)

Ksv = Kq x T0

bioluminescent reactions

Light-emitting reactions arising from a LIVING organism (e.g. firefly/jellyfish) Basically chemiluminescent observed in NATURE

Bioluminescence systems

Luciferase enzyme catalysed oxidation of luciferins. Principle of this system: - Involves a light-emitting molecule (luciferin) + enzyme (luciferase)

Self-Absorption/Inner filter effect

Occurs when emission intensity overlaps with absorption peak

Good reference compounds for fluorescence

Perylene diimides and rhodamines

What is quenching?

Process which decreases fluorescence intensity of a given substance

Chemiluminescence

Produced when a chemical reaction YIELDS an electronically excited species (product) that emits light as it returns to its ground state - Release of energy from: CHEMICAL REACTION - Using synthetic compounds/highly oxidized species Formation of electronically excited reaction product (by oxidation) - Direct A + B --> C* + D C* --> C(ground) + hv C is the new product, and it is DIRECTLY EXCITED instead of starting from ground state - Indirect A+B+F --> C+D+F* F* --> F + hv Unlike C, F has to start from ground state before getting excited. SOOOO it is NOT DIRECT

How is QY of fluorescence calculated by?

Rate constant at which fluorescence occurs (Kr) / (Kc + sum of all possible rate constants for deactivation of excited state)

When you assume first order kinetics, what are you assuming?

Reaction of peroxide with base is close to first order at the concentrations used for generating chemiluminescence.

Kasha's rule

The excited molecule first reaches the LOWEST vibrational level of S1 --> and THEN photon emission occurs always from this state TO any vibrational level of the GROUND (S0) state (Fluorescence)

Consequences of Kasha's Rule

The rate constant of internal conversion and vibrational relaxation from the higher lying electronic levels is significantly greater than the emission rate constant back to the ground state from those levels Emission and absorption spectra = mirror images

Define natural lifetime (fluorescence)

Time at which probability of emission having occurred is a certain percentage when there are NO other ways for amolecule to relax other than emitting a photon

In what case is sigma (2pz) lower in energy than pi (2py) and pi (2px)?

When average atomic number is more than or equal to 8 (e.g. O2)

Fluorescence

electron absorbs light at one wavelength and then emits light at a LONGER wavelength (radiative relaxation)

How to calculate natural fluorescence lifetime?

observed fluorescence lifetime/QY fluorescence

Chemiluminescence

the emission of light from a CHEMICAL reaction