Optical Flashcards
Whare are chromophores?
Functional groups which give rise to observed transitions
UV-VIS
Why does spectrum broadening happen?
- large number of overlapping vibronic transitions
- transitions broadened by collisions with solvent
Beer Lambert Law
What are the fundamental limitations?
3 points
- Invalid at high concentrations
- Refractive index depends on concentration
- Extinction coefficient depends on refractive index
Why is Beer-Lamberts law invalid at high concentrations?
Aggregation of molecules affects extinction coefficicient
Beer-Lambert Law
What type of deviation does high sample concentration lead to?
Positive
Beer-Lambert Law
What are instrumental limitations?
2 points
- Stray radiation reaching detector without passing through sample
- Polychromatic radiation, as BL assumes monochromatic radiation
Beer-Lambert Law
What type of deviation does stray radiation lead to?
Negative
Define bandwidth
The full-width of the radiation at half of its maximum intensity
Beer-Lambert Law
Why do you measure absorbance at the top of the absorbance peak
BL assumes monochromatic radiation, spectrometer has finite bandwidth. Effect is minimised if the value of epsilon is constant over bandwidth (top of the abs peak)
Describe the procedure for quantitative analysis using Beer-Lamber Law
4 points
- Measure absorbance and apply BL law
- Construct a calibration curve
- Linear regression of absorbance versus concentration to give calibration equation
- Substitute sample’s absorbance into calibration equation to give analyte concentration
How is qualitative analysis carried out using UV-VIS?
- Use characteristic absorption spectrum of chromophore to identify compound
- Compare to the library of digital compounds
- Use of derivative spectra can improve discrimination between similar spectra
- UV-vis spectra tend to be broad and unstructured – not “fingerprints”
- Use PCA for multi-component mixtures
Define Luminescence
Emission of light by electronically excited molecules
Define Fluorescence
Molecule excited by absorption of light
Compare and contrast Fluorescence and Phosphorescence
F: spin allowed and very short lived
P: Spin forbiddena and long lived
What is a singlet electronic state?
When all the electron spins are paired in the molecular electronic state and the electronic energy levels do not split when the molecule is exposed into a magnetic field
What is a triplet electronic state?
Two electrons in different molecular orbitals have parallel spins
Fluorescence
What is vibrational relaxation?
- Collision with solvent molecules -rapid loss of vibrational energy to solvent as heat.
- Moving down one energy level within the same state
Fluorescence
What is Excitation (absorption)?
Promotion of electrons into S1/2 etc into the same energy level
Fluorescence
What is fluorescence?
Transition from lowest vibrational level(s) of S1 state to S0
Fluoresence
What is internal conversion?
Conversion of electronicenergy to vibrational energy S1 -> S1
What is Kasha’s Rule?
Fluorescence always from lowest vibrational levels of S1 state to any level of S0
Fluorescnce
What is intersystem crossing?
Transition between states of different multiplicity (S1 to T1/ T1 to S0)
Fluorescence
Fate of radiactive decay
3 points
- Radiative
- Non radiative
- Intramoleculay decay
Fluorescence
What is radiative decay?
3 points
- Energy lost by emission of a photon
- Fluorescence S1 to S0 spin allowed
- Phosphorescence T1 to S0 spin forbidden
What is on the axes of Stern-Volmer plot?
F0/F vs [Q]
What is the gradient and intercept of Stern-Volmer plot?
grad: K_SV (Stern-Volmer constant)
intercept: 1
Fluorescence
What is the innter filter effect?
2 points
- If concentration is too high maximum fluorescnce happens in the middle of the sample
- This results in a distorted spectrum, as intensity in the middle of cuvette is reduced.
Fluorescence
How can the inner effect be prevented?
Absorbance should be < 0.1 at excitation wavelength
Fluorescence
What is spectrum reabsorption?
3 points
- If absorption and emission spectra overlap, and concentration is too high fluorescence is reabsorbed as it passes through solution
- Fluorescence intensity is reduced
- Emission spectrum distorted
What is a fluorophore?
Fluorescent molecule or group
- What groups do flurophores contain?
- Which transition does it allow?
- Aromatic groups
- pi to pi*
List advantages of fluorescence over UV-VIS
5 points
- High detection sensitivity
- High sensitivity
- Use as probe
- Sensitive to molecular environment
- Sensitive to molecular interactions
Which 3 characteristic parameters are responsible for high sensitivity of fluorescence over UV-VIS
- Excitation wavelength
- Emission wavelength
- Lifetime
List disadvantages of fluorescence over UV-VIS
5 points
- Intensity of fluorescence is not a direct measure of concentration
- Intensity depends on experimental & instrumental factors
- Intensity depends on quantum yield
- Requires careful production of calibration curves
- Ratiometric measurement advantageous
What are the experimental and instramental factors that affect fluorescent intensity?
4 points
- Intensity of excitation source
- Efficiency of collection of fluorescence
- Inner filter effect
- Reabsorption
Fluorescence
What is quantum yield affected by?
2 points
- Sample conditions (pH, temp, quenching)
- Intermolecular interactions
IR
How does a FTIR work?
4 points
- Transmittance is measured (like UV-VIS)
- An interferometer is used instead of monochromator
- All IR frequencies incident on detector simultaneously
- Fast spectrum acquisition
IR
How does an interferometer work?
- Interferometer produced signal which has all the infrared frequencies encoded into it
- Fourier transform converts intensity versus time to intensity versus frequency
