Spectroscopy Flashcards
UV spectrum
Visible light = 340nm - 600/800nm
UV light = 200nm-340nm
Absorbance is preferentially measured in the visible light range because it uses plastic cuvettes and tungsten lamp, making it cheaper
Absorption spectroscopy
Uses light absorbed at very distinctive wavelengths, giving peaks
Peaks are generated when a photon of light is absorbed, exciting an electron
Photoluminescence
Emission of light following absorption of photons
When a compound absorbs light, an electron in the singlet ground state is excited to the singlet excited state
When the electron drops down to ground state, it releases energy, causing emission of light
Stokes shift
The photon of light emitted from a compound is at a higher wavelength and lower frequency than the photon of light absorbed by the same compound
This because some energy is lost due to collision with other molecules in the system
Fluorescence
Based on the principle that a photon of light is emitted from the singlet excited state to the singlet ground state
Fluorescence decays rapidly once the source of excitation is removed
Detection of fluorescence
A fluorimeter is used to detect fluorescence
Measurement is made at a 90 degree angle from the direction of the light source to avoid contamination of the result
Advantages of measuring fluroescence
- Helps identify specific compounds
- More sensitive than absorbance alone
- Determines the environment of the compound
- Can measure molecular distances
FRET
A special technique used to gauge the distance between two chromophores, allowing distance to be measured on a molecular level
Can quantify distances 1-10nm
Quantum yield
The fraction of excited state molecules returning to the ground state by fluroescence
Fluorescence quenching
Any process that decreases fluorescence intensity
Uses of FRET
- Examines protein-ligand interactions
- Examines protein-protein interactions
- Biochemical assays
- Identification of conformational changes
FRAP
Fluorescence recovery after photobleaching
A method of determining the kinetics of diffusion in living cells
GFP (green fluorescent protein) is distributed evenly throughout the cell
A laser is then used to burn out the fluorescence
GFP then diffuses to fill the burned area and hence rate of diffusion can be measured
Confocal microscopy
A method of fluorescence microscopy
Uses a laser as the excitation light source, allowing optical slicing so a 3D picture of the cell can be revealed
Biological stains
- DAPI = stains nuclei blue
- Fluorescein
Green fluorescent protein
A small protein of 150 amino acids
The chromophore gives the molecule fluorescence and is made up of:
- Ser65
- Tyr66
- Gly67
It is a B-barrel structure filled with a-helicies which prevents water from entering the structure, which would quench the chromophore
