Spectroscopy Flashcards
What is light?
An electromagnetic wave that is made up of an electric field and a magnetic field that oscillate perpendicular to each other
What equation involves wavelength?
Energy (E) = hc/λ
h = Planck’s constant
c = speed of light
λ - wavelength
OR
λ = c/v
v = frequency (s-1 or Hz)
Define the types of energy radiation?
High energy radiation -
short wavelength and high frequency
Low energy radiation -
long wavelength and low frequency
What is amplitude?
The maximum value the electric or magnetic vector can have
What happens when two waves are superimposed on each other?
The resultant wave = the sum of the waves
180 degree shifted - cancel out
90 degree shifted - the wave will form in the middle of the peaks
How does light interact with molecules?
Scattered - the direction of propagation changes
Absorbed - energy is transferred to the molecule
What happens when energy is absorbed?
When the electromagnetic radiation is absorbed and the molecule becomes excited
The area that absorbs the energy is the chromophore
The energy acquired moves and electron to another orbital - this is called a transition
Ground state -> first excited state
What do excited molecules possess?
Discrete amounts of energy = quanta
Amounts - electron energy levels
Vibrational energy levels - superimposed on electron energy levels
What can we measure as a graph?
Probability of photon absorption vs wavelength
This is called the absorption spectrum
What is the beer lambert law?
A = εcl
A - absorbance
ε - molar extinction coefficient
c - concentration
l - length
What is significant about the beer lambert law?
Where the absorbance appears proportional to concentration the beer lambert law is obeyed
If concentration is increased it can lead to: oligomerisation, aggregation and denaturation
How do we measure absorbance?
Using a spectrophotometer
Light source Monochromator - provides selectivity of wavelength Sample holder Detector - photocell Recorder
The spectrophotometer needs to be zeroed
What factors affect the absorption of a chromophore?
Polarity
pH
Orientation
How does polarity affect absorption of a chromophore?
A polar chromophore placed in a polar solvent can lead to:
Hypsochromic shift - shorter wavelengths (more blue)
Non bonding orbital -> pi anti-bonding orbital
bathochromic shift - longer wavelength (more red)
pi bonding orbital -> pi antibonding orbital
How does pH affect the absorption of a chromophore?
pH can determine the ionisation state of a chromophore that is ionisable
This can shift the equilibrium to the right sue to: more delocalised electrons and increased molar extinction co-efficient (bathochromic shift)
How does orientation affect the absorption of a chromophore?
The geometric features of nucleic acids can lead to:
Hypochromism - a materials decreasing ability to absorb light
Hyperchromism - a materials increasing ability to absorb light
How do we correct scattering?
This is needed if particles are in suspension and not solution
- as it leads to an artificially high absorbance
To correct: measure points away from the max wavelength in order to extrapolate the linear region
What is a reporter?
A ‘non-natural’ molecule added if the molecule has no chromophore
It has to bind at a single site and can’t affect the function or interaction
What information can be obtained from UV-visible spectroscopy?
The concentration of a substance
An assay of certain chemical reactions
Identification of materials
Solvent perturbation
What is solvent perturbation?
If you change the solvent of nucleic acids from H2O to 50% of D2O
It can change the mono-nucleotides but not the base pairs
This is due to N-D having a greater reduced mass, altering the vibrational levels and disallowing the ability to hydrogen bond
What can happen after absorption of a photon?
The energy can be reemitted as heat
OR
Emit a lower energy photon seen as fluorescence
How does fluorescence work?
A photon is absorbed by a fluorophore and the excited electrons move to a higher energy level (the first excited state) in a transition
When the excited molecule relaxes a new low energy photon is emitted
The emission spectrum is shifted to a longer wavelength compared with the excitation spectrum (Stokes shift)
How is probability of fluorescence described?
The quantum yeild (Q)
Q = ratio of emitted photons to absorbed photons
Q is determined by:
The properties of the molecule itself
The local environment of the molecule
How else can energy be transferred is not heat/fluorescence?
If the vibrational levels of the excited and ground states overlap, energy can be transferred from the higher energy level to the lower – without re-emission of a photon, therefore no net transfer of energy
= nonradiative transfer
How is fluorescence measured?
Using a fluorimeter
Commonly measured at 90º or as backscatter because fluorescence is emitted in all directions
What type of fluorescence is measured?
Intrinsic fluorescence: an intrinsic fluorophore is one contained within the macromolecule
an amino acid (W, Y or F) – (Mainly tryptophan as it has the highest yield – most commonly studied)
Extrinsic fluorescence: an extrinsic fluorophore is one added to the macromolecule
It should be attached at a single site and not change its properties
Each fluorophore can be distinguished by its excitation and emission wavelengths
What environmental impacts can we use to alter fluorescence readings?
Polarity Quenchers Temperature pH Ligand binding
What are quenchers?
Quenching a process which decreases the fluorescence intensity of a given substance
Tryptophan quenchers: iodine, nitrate, cesium and acrylamide
A quencher only works if the fluorophore is on the surface not buried
What is FRET?
FRET - Förster Resonance Energy Transfer
If the emission of one fluorophore overlaps with the absorption peak of a second fluorophore the energy can be transferred between them in a nonradiative process
