Fluorescence Spectroscopy Flashcards
What is a non-radiative transition?
Prior to emitting the photons and returning to the ground state, excited molecules may undergo non-radiative transitions (e.g., release heat)
What is fluorescence?
One type of luminescence (light emission from a molecule)
a type of photoluminescence
the phenomenon that a material emits light at one wavelength when you illuminate it with another
What are the 3 types of luminescence?
- Photo-
- Chemi-
- Electro-
-luminescence
Define: photoluminescence
Light energy, or photons, stimulate the emission of a photon
Absorption and emission bands […]
often appear as image and mirror image
Why is emission light a different wavelength?
- Some energy is lost
- In emission you will see a ‘red-shift’
- No energy change, frequency change, or wavelength change between green light absorption and emission.
Compare the excitation and emission spectra.
- Absorbs light at lower wavelengths (excitation)
- Emits light at longer wavelengths (emission)
The stokes shift describes the energy difference between the absorption and emission spectra.
Define: chemiluminescence.
chemical energy stimulates the emission of
a photon
Define: electroluminescence
electrical energy or a strong electric field, stimulates the emission of a photon
What is ‘Stokes shift’
- The energy difference between absorption and emission spectra.
Stokes shift refers to the difference between the wavelength of maximum absorption and the wavelength of maximum emission in a fluorescent molecule.
What are the advantanges of fluorescence spectroscopy?
- Very sensitive: 1000x more sensitive than absorption spectroscopy
- Non-destructive: sample remains intact, in contast to some methods like mass spectrometry
- Highly selective: fluorescence experiment involve two analytical wavelengths—the excitation and the emission wavelength. Only compounds that absorb the excitation light and emit light at a certain wavelength are detected.
- Very informative: provides qualitative and quantitative information (Detection, Characterization, Sample environment [etc. temperature, pH])
What signals are provided by fluorescence that are used in quantification?
- Number of excited compounds: The fluorescence intensity is proportional to the number of excited molecules in the sample.
- Fluorescence quantum yield (how likely a molecule in the excited state starts to emit light
What is fluorescence intensity proportional to?
What is the equation for quantification by fluorescence?
Fluorescence intensity is proportional to
concentration for very diluted solutions (A≤0.05)
Why is fluorescence used for quantification of very diluted solutions?
- Fluorescence intensity is proportional to concentration for very diluted solutions (A≤0.05); the relationship is linear
Define: Fluorophore
- A fluorescent chemical compound that can re-emit light upon light excitation.
- Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several Pi bonds
Give examples of fluorophores. [5]
- Organic dyes: chlorophyll (natural organic dye) or fluorescein (synthetic organic dye)
- Biological fluorophores: green fluorescent protein (GFP)
- Quantum dots : nanocrystals (2-50 nm), semiconductor particles having optical and electronic properties.
- Intrinsically fluorescent: chlorophyll and the amino acid residue tryptophan (Trp)
- Molecules synthesized as stable organic dyes or tags to be added to non-fluorescent systems
Give an example of a natural fluorophore.
- Chlorophyll (a & b)
- Absorbs red and blue wavelengths
- Reflects green
- Fluoresces in the red wavelengths
What are quantum dots?
- When excited, emit fluorescence at a wavelength based on the size of the particle
- Smaller quantum dots emit higher energy than larger quantum dots
Widely used probe.
Drawbacks: cell toxicity; expensive
What are the unique optical and electronic properties of quantum dots? [3]
- Size-tunable emission
- Extreme brightness
- Resistance against photobleaching
Smaller quantum dots emit higher energy than larger quantum dots.
Smaller quantum dots emit higher energy than larger quantum dots.
True or False?
True.
Larger quantum dots emit higher energy than smaller quantum dots.
True or False?
False.
Smaller quantum dots emit higher energy than larger quantum dots.
What are drawbacks of quantum dots? [2]
- Cell toxicity (intrinsic property; often synthesized from metals)
- Expensive
Widely used probe.
List a few of the categories of fluorescent molecules and materials.
Define: quenching
Refers to any process which decreases the fluorescence intensity of a given substance
Molecular oxygen, iodide ions and acrylamide are common chemical quenchers. The chloride ion is a well-known quencher for quinine fluorescence.
Give examples of quenching processes. [4]
- Excited state reactions
- Energy transfer
- Complex-formation
- Collisional quenching
Molecular oxygen, iodide ions and acrylamide are common chemical quenchers. The chloride ion is a well-known quencher for quinine fluorescence.
Fluorescence quenching is […]
Heavily dependent on pressure and temperature
Molecular oxygen, iodide ions and acrylamide are common chemical quenchers. The chloride ion is a well-known quencher for quinine fluorescence.
What is FRET?
Förster resonance energy transfer
A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling.
The efficiency of FRET is […]
The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
What are the 3 requirements for FRET to occur?
- Spectral overlap
- Distance <10 nm
- Correct orientation
Give an overview of fluorescence instrumentation.
Describe differences in the excitation monochromator that are used in fluorescence spectroscopy.
What are the applications of fluorescence spectroscopy?
- Quantitative analysis
- Qualitative analysis
- to study interaction between molecules
- to study molecule structure
- to study the effect of environmental factors on molecule structure
Two approaches
- To follow intrinsic fluorescence of analyte
- To use a fluorescent probe
When may intrinsic fluorescence not be a suitable approach?
- Compounds may not fluoresce
- Interaction between aromatic residues (hard to interpret spectrum)
A fluorescent probe in this case can be helpful (extrinsic fluorescence)
Describe characteristics of the ideal fluorescent probe. [5]
- Small, does not perturb molecule confirmation of interest
- Has a specific binding site
- Forms a stable complex
- Has a simple emission spectrum
- Effected by environmental factors (e.g., pH, temp., etc.)
Give two examples of ideal fluorescent probes.
- ANS (binds hydrophobic site of protein)
- cis-Parinaric acid (study the hydrophobicity and foaming characteristics of food proteins)
Desribe the use of probes for studying microplastics.
The fluorescence of quantum dots could be quenched by […] because of […].
The fluorescence of quantum dots could be quenched by aqueous nitrite because of the electron transfer between the electron-donating functional groups on the surface of the carbon dots and the electron-accepting nitrous acid.
