Spectrometry Flashcards
What is the difference between spectroscopy, spectrometry and spectrophotometry?
How could you quantify an absorbance spectrum?
What are the main 7 components of a spectrophotometer?
What does a monochromator consist of?
Spectroscopy: study interaction matter with light
Spectrometry: method to acquire spectrum
Spectrophotometry: spectrometry in UV-visible-near infrared region
Measure amount of light absorbed at each wavelength
Source (halogen lamp/tungsten filament), entrance slit, dispersion device (prism/diffraction grating), exit slit (selects one of the split wavelengths), sample, detector (measure light intensity to calculate absorbance), amplifier/readout (computer)
Entrance slit & dispersion device
How does a prism disperse light?
How does a diffraction grating disperse light?
What are the benefit of each dispersion element?
Different wavelengths travel at different speeds, so longer red wavelengths are refracted less than shorter violet ones.
The groove period is on order of wavelength of interest- splits light into several beams. The spectral range depends on the groove spacings.
Prism = transmittance around 100% as efficient in visible light range
Grating = wavelength dispersion essentially constant
How is the specific wavelength of light isolated/selected?
What are the 3 types of cuvettes? Which one is better and why?
What can be used as a detector and what does it do?
How could you find the concentration of an unknown sample?
With a narrow exit slit
Quartz & polystyrene/plastic, glass.
Quartz works from 150-800nm wavelength compared to glass & plastic that only work after 300nm-800nm.
Photomultiplier/photodiode- convert stream photons into stream of electrons (into an electrical current)
Plot the absorbance values on a standard curve & read off to find the unknown concentration
What is the typical units of an extinction coefficient?
What is an isosbestic point?
What can it be used to calculate?
M-1cm-1
Point at a particular wavelength when 2 chemical species have the same extinction coefficient/so the same absorptivity regardless of their ratios.
Calculate the total amount of the 2 species combined but not their individual concentrations.
What is fluorescence?
What is different about the light emitted to the light absorbed?
Why are fluorescent methods sensitive?
What 3 amino acids cause intrinsic fluorescent proteins?
What can fluorescent compounds be used for?
Photons are absorbed by a molecule & after a time part of the energy is re-emitted as light with a lower energy.
Light emitted in fluorescence is at lower energy/longer wavelength.
Low background signal & ability to detect small numbers emitted protons.
Tryptophan, tyrosine, phenylalanine
Covalent link to proteins, attach to antibodies, label filaments to visualise motions
How can GFP allow proteins to be visualised/located?
What is GFP’s structure? Why is it significant?
What are the 3 amino acid residues involved in the GFP chromophore? How can the colour of GFP be changed?
GFP gene is spliced into the gene that codes the interested protein- when protein expressed GFP is covalently attached so the GFP folds into its active fluorescent form.
Anti-parallel barrel structure incasing the fluorophore to protect it from quenching by water.
Gly67, Tyr66, Ser65
By replacing these residues with others
How can GFP allow proteins to be visualised/located?
What is GFP’s structure? Why is it significant?
What are the 3 amino acid residues involved in the GFP chromophore? How can the colour of GFP be changed?
GFP gene is spliced into the gene that codes the interested protein- when protein expressed GFP is covalently attached so the GFP folds into its active fluorescent form.
Anti-parallel barrel structure incasing the fluorophore to protect it from quenching by water.
Gly67, Tyr66, Ser65
By replacing these residues with others
Why is fluoresence measured at a right angle?
What is Stokes shift?
What are the main components of a spectrofluorimeter?
If the peak absorbance of GFP is at 395nm and peak emission at 509nm, what would you set the excitation and emission monochromator?
At a right angle to the exciting light beam in a fluorimeter- avoid detection of transmitting and reflected incident light.
Different between maxima of absorption & emission (longer wavelength) spectra
Xenon arc lamp, excitation monochromator, sample, at 90degrees: emission monochromator, photon detector
Excitation = 395nm, Emission = 509nm
How can fluorescent spectroscopy be made most accurate?
Why is fluorescent spectroscopy very sensitive? 2 answers
How can you overcome these?
What is directly proportional to the intensity of the fluorescent light? Why is it important to produce a calibration curve?
What is the quantum yield?
What does it depend on and how can it be used?
What can fluorescence be used for?
At low fluorophore concentrations
Stokes shift- wavelength of emitted light is different to excited light. Fluorescence is non-polar and is emitted in all directions.
Set the emission and excited monochromators separately. Place the detector perpendicular to the excitation pathway (to minimise incident beam)
Concentration of the fluorescent molecule- factors can alter a linear relation
Absolute measure of fluorescence- ratio of photons emitted and photons absorbed by a fluorophore- it’s dimensionless.
Depends on nature of environment and the fluorophore & its proximity to other molecules-
(so can be used to monitor binding of molecules/structural changes in a fluorophore, or also comparison between 2 related samples.)
Detecting DNA on gels, labelling proteins with chemicals and antibodies, protein dynamics, use intrinsic proteins to monitor structural changes/binding of ligands, measure enzyme rates, tagging proteins to monitor expression levels & locations etc
Molecule absorbs a photon and electrons are excited from S0v0 the S2v2 electronic level in an excited vibrational state. What is the step after this? How fast is it?
Internal conversion is the step afterwards. What is it and what energy is released?
The electron stays at this state for some time & then 1 of 3 events occur: what are they?
T1 to S1 and T1 to S0 - which is forbidden? Why is it forbidden?
How can molecule in T1 state return to SO state?
What happens to the extra energy from the absorbed photon in fluorescent & non-fluorescent molecules?
Molecule returns to S2v0 ground vibrational state & energy given off as heat = vibrational relaxation. Very fast.
Change of electron from an electronic state to another electronic state of the same spin type (so S2v0 to S1v0). Very fast & given up as heat.
When molecule returns to S0v0 can either:
Fluoresce & energy lost as light
Internal conversion & energy lost as heat
Intersystem crossing & heat- electron changes spin state so S1 to T1
T1 to S1 is not forbidden, but S1 to S0 meaning transition is slow (seconds instead of milliseconds) Forbidden as involve transitions in spin and orbital state.
Return via phosphorescence (light) or intersystem crossing (heat)- very slow process
Fluorescent- used to emit light
Non- dissipate by vibrating faster/giving off heat & return to lower energy state
How can you achieve an emission spectrum?
What about an excitation spectrum?
What can they be used for?
How is fluorescence intensity different to absorbance?
Vary the emission monochrometer. Don’t vary conditions/wavelengths of excited light.
Vary excitation monochrometer but under conditions where wavelength of emitted light doesn’t change.
Emission: colours emitted by molecule
Excitation: tell what colours cause molecule to go into excited state- same wavelength to absorbance
FI depends on response of instrumental components as a function of wavelength- different instruments = different intensity spectra
How can you achieve an emission spectrum?
What about an excitation spectrum?
What can they be used for?
How is fluorescence intensity different to absorbance?
Vary the emission monochrometer. Fix wavelength of excited light
Vary excitation monochrometer but under conditions where wavelength of emitted light doesn’t change.
Emission: colours emitted by molecule
Excitation: tell what colours cause molecule to go into excited state- same wavelength to absorbance
FI depends on response of instrumental components as a function of wavelength- different instruments = different intensity spectra
Since excitation is parallel to absorbance and emission is related to fluorescence, what is expected of the two graphs?
What happens instead? And what are the 2 effects?
What can fluorescence be used for?
What happens when fluorophores move into a less polar environment? What about increase temperature & polar environment?
Expected to overlap- as So to S1 transition is opposite to So to S1 absorbance
Don’t completely overlap (results in Stokes shift)- due to vibrational splitting & solvent effects
Quantitate material (absorbance)- careful due to equipment. And study protein folding/ligand binding with environmental change (intrinsic proteins) with spectral shift
Emission spectrum moves shorter wavelengths. Decrease in quantum yield.
What is the inner filter effect?
What is it caused by?
What can it lead to?
How can you overcome this?
What type of organic molecules typically fluoresce?
What are the problems with Tyr in a protein?
When high concentration of fluorescent molecules are absorbed in cuvette before hitting molecule of interest (deviates from linear C vs F, so concentration decreases causing curve)
Absorber in the cuvette
Artifacts: wrong fluorescent conc estimate and false binding conclusions
Reduce concentration of the sample
Conjugation systems, rigid, planar (proteins)
10x less fluorescent than Trp, and FRET (excited energy transfer from Tyr to Trp)
What are the 2 types of non-covalent fluorescent probes? How are these detected?
How do covalent fluorescent probes work? What are their uses?
How could you monitor conformational change of a protein?
How could you monitor ligand binding?
How does Trp fluorescence change in hydrophilic (exterior) or hydrophobic environments (interior)?
Ethidium bromide (detect DNA on gels) and diphenylhexatriene (measure physical state of lipid bilayers & detect micelle formation). Increase in fluorescence in non-polar/hydrophobic environment
F group attaches to chemically reactive X group (SH (cys) and NH2 (lys) reagents) that reacts with protein. Probe local environments (amino acids), monitor conformation change & detect location molecules.
With Trp fluorescence or label protein at Cys residue with F probe
Change in protein fluorescence or change in ligand fluorescence
Hydrophilic = red shift/longer Hydrophobic = blue shift/shorter
