Flourescence Spectroscopy Flashcards
Are florescence spectra of molecules ever a mirror image of an absorption spectrum? Why?
Spacings of the energy levels for the first excited state are similar for absorption and fluorescence processes
This implies that the florescence spectra of molecules is near to mirror image of the absorption spectrum, BUT in reality this is only the case for simple molecules
Y axis and X axis of fluorescence spectrum?
Y axis = fluorescence emission (arbitrary units)
X axis = wavelength (nm)
Do you need to differ things from different machines?
Yes because different machines will have different units
Intensity is proportional to…
The amount of photons emitting that the detector picks up
Where does the excitation wavelength come into things on the spectra shape)
The shape of this kind of spectrum does NOT depend on which excitation wavelength was used, whereas the intensity of emission does depend on the excitation wavelength
What does the excitation spectrum allow for?
Allow/helps identify suitable wavelengths that can be used to excite the fluorophore
When is the excitation spectrum equal to the absorption spectrum?
For dilute solutions
It is a plot of intensity of emission as a function of wavelength
How fast is fluorescence?
Nano seconds
What does the process of luminescence involve?
Excitation: electrons excited to a higher energy state by light absorption
Luminescence: electrons return to ground state and light is then emitted with less energy - some energy is lost
How is energy lost when electrons return to the ground state?
Intersystem crossing
Internal conversions
Relaxations from vibrational and rational levels
What is a Jablonski diagram?
Energy diagram that describes the process of photon emission
How does fluorescence happen again and again and repeat?
Constant S0–>S1–>S0 allows this to happen
What is intersystem crossing?
Molecules relax via non-radiative transition to T1
Requires spin orientation to change
What is internal conversion?
Radiation-less transition but vibrational levels need to match
In fluorescence, how does energy get lost most commonly from relaxation?
Return from excited singlet state to ground state. Does not require change in spin orientation
Lifetime=nano seconds
What is phosphorescence
Return from the triplet excited state to the ground state
Requires a change in spin orientation
Usually forbidden but does happen
Longer lifetime than fluorescence- milliseconds rather than nanoseconds
Generally occurs at longer wavelengths than fluorescence because energy difference between S0 and T1 is smaller
What is a fluorophore?
A chromophore that can emit fluorescence
Describe the excitation process of fluorescence in detail
Amount of time this takes?
Upon light absorption, a chromophorre that was in the lowest vibrational state (V0) of the ground state S0 is excited to some higher vibrational level (e.g. V1 or V2) of the S1 or S2 state
10^-15 seconds
Describe the vibrational relaxation process of fluorescence in detail
Non-radiative relaxation takes place until the lowest (relaxed) vibrational level of S1 is reached.
Molecule may undergo conformational change or react with the environment to achieve this
Time: takes 10^-12 seconds
Describe the third process of fluorescence in detail, after vibrational relaxations occur
The molecule then relaxes from this lowest vibrational energy level of the excited state S1 to one of the vibrational energy levels of the ground state. In doing so, fluorescence is emitted
Time: 10^-9 secs
What are the following: S1 S2 T1 S0
S1 = 1st excited singlet state S2 = 2nd excited singlet state T1 = 1st excited triplet state S0 = ground state
What is Stoke’s shift?
Difference between where you apply energy and the maximum peak for emission
I.e.
The difference in wavelength between absorption and emission
In Stokes shift, how is some of the absorbed energy lost?
Due to processes that happen in the excited state limetime e.g.
- vibrational relaxation
- collisional quenching
- energy transfer to other molecules (FRET)
- intersystem crossing
- photobleaching
In Stokes shift, if some of the energy is lost during the excited state lifetime, what can we deduce about the wavelength of light emitted by fluorescence
The wavelength of light emitted by fluorescence is higher than the light that was absorbed
Describe the relationship between:
Speed of light, frequency, wavelength
And
Energy and frequency
c=f(wavelength)
E=hf
Where h= Plank’s constant and c is the speed of light
Is it good for Stokes shift to be small or big and why?
The larger the better, because you can detect more sensitively without detecting input radiation (incidence light)
25nm shift is good
How do you perform measurement of fluorescence?
Adjust the concentration: 10 to 100 times less concentrated than those used in absorption spectrometry because fluorescence is very sensitive
Transfer solution to a 4-sided cell
Make sure there are no solid particles, as these cause light scattered
Ideal absorption of solution to be put in 4-sided cell?
<0.05
Is instrument to instrument variation a thing with this kind of measurement?
Yes. It is so significant that intensity from one machine is very different to that of another
Thus fluorescence is measured in arbitrary units and quantitive analysis can only be done by comparing reading with a reference or a calibration curve obtained on the same instrument
Advantages of using fluorescence?
- very sensitive (ug to ng, depending on quantum yield)
- higher selectivity than UV-Vis
- relatively inexpensive (more expensive than UV-Vis but less expensive than MS and NMR)
- can be applied in many cases without separation step (e.g. Chromatography)
Limitations of fluorescence
- not all drugs are fluorescent
- use of standards normally required in pharmaceutical analysis (arbitrary units)
- changes in conditions can affect fluorescence properties
Describe the instrumentation of fluorescence spectroscopy
Lamp or laster (Xe arc lamp or laser) Excitation Monochromator Cell holder (4-sided and clear) Emission monochromator (at 90 degrees to excitation one) Detector
Why is the excitation monochromator and the emission monochromator at 90 degrees to each other?
So excitation light doesn’t hit the detector (incident light)
Fluorescence is a glow and goes in all directions
In fluorescence spectra, how many times is the sample excited and at what wavelengths?
When recording a fluorescence spectrum, the sample may be excited at a single wavelength (excitation wavelength) and the emission recorded at several wavelengths.
What must be considered when there are 2 molecules with different absorption spectra i.e. Mixed fluorophores?
When there are 2 molecules with different absorption spectra, it is important to consider where a fixed wavelength excitation should be placed
Describe a method used whereby there are mixed fluorophores
Why is it good?
Excite at different wavelengths
- distinct peaks
- if overlap–> can see both
This method can also be important to minimise fluorescence from impurities by selecting an excitation where impurity cannot absorb light
Name the main factors that can affect fluorescence intensity
Inner filter effect pH Photobleaching Temperature Viscosity Presence of oxygen Structural solvent
Describe what photobleaching is and how it occurs
Too much incident radiation can irreversibly degrade the fluorophore and cause loss of fluorescence
This is photobleaching and occurs with lasers
How does the presence of oxygen affect fluorescence intensity?
High concentration of oxygen quenches fluorescence through collisions
For example, Copper (Cu2+) ions quench fluorescence by complexation-static quenching
How does viscosity and temperature affect the fluorescence intensity?
Viscosity and temperature have opposite effects
As molecules move more freely (low viscosity or high temp) there is more chance of intermolecular collisions (involving solvent molecules) happening.
This causes loss of energy or deactivation of excited state by nn-radioactive means and thus lower fluorescence
How does pH affect the fluorescence intensity?
Fluorescent drugs with ionisable groups can be sensitive to pH
Barbiturates only fluoresce in the di-anionic form and phenol fluoresces at pH 7, when it is an anion
What is the inner filter affect?
If you plot intensity(y) against concentration (x) the plot forms almost a U-shape. This means at certain intensities, the intensity could be at two different concentrations.
At high concentrations of drug, fluorescence intensity reaches a plateau and as conc increases further, the intensity will decrease due to inner filter effects, in which the ground state molecules absorb (present in abundance) the fluorescence emitted by the excited molecules.
This explains why we dilute the sample as it gives a linear range
How is fluorescence used in BP?
Used as a standard for quantitive analysis
Fluorescent light emitted by the substance examined in relation to that emitted by a standard.
Dissolve substance in solvent.
Set excitation wavelength. Measure the intensity of the emitted light at an angle of 90 degrees to the excitation beam.
How is the BP method used as a formula?
State the formula that gives the concentration of the solution to be examined
When is this method valid?
Cx=(IxCs/Is)
Where:
Cx = concentration of solution to be examined
Ix= intensity of light emitted by solution to be examined
Cs= concentration of standard solution
Is=intensity of light emitted by standard solution
Method only valid if fluorescence is linear with concentration (i.e. No inner filter affect)
Describe the influence of solvent choice on fluorescence
Polar fluorophores are especially sensitive
Interaction with solvent molecules can offer a non-radiative pathway for energy loss
Why would you choose Fluorescence over UV?
Example: ethyinylestradiol tablets
UV not good as excipients absorb too and the drug at a low level (mg)
In this assay, use is made of pH affect to correct for background fluorescence from excipients
Addition of NaOH ionises the phenolic group to make it non-fluorescent
Affect of rigidity on fluorescence?
Rigidity affects fluorescence e.g. Formation of chelate (BP limit test for aluminium)
Effect of substituents that can affect resonance stability changes on fluorescence?
Electron-withdrawing groups lower fluorescence (e.g. COOH, Halide ions, nitro group) while electron-donating groups raise fluoresce (e.g. NH2, OH)
What is derivatisation and how is it used in fluorescence spectromtery?
You add something else onto the molecule you are testing
E.g.
-Some drugs can be made to become fluorescent by chemical treatment (e.g. Digitoxin treated with H2SO4 shows strong fluorescence)
-drugs can also be labelled with specific fluorophores that are reactive towards a certain functional group (generally amines, COOH or SH)
Protein/peptide drugs contain what fluorescent groups and how do these groups react after protein folding?
Peptide/protein drugs may contain tyrosine and/or tryptophan that are fluorescent.
The fluorescence of such drugs changes on folding/defolding (denaturisation) as a result of solvent access and rigidity change
Problem with derivitization of molecules for fluorescence spectroscopy?
Difficult to know how selective the group is and may have to add a separation step
