UV-Vis and fluorescence spectroscopy Flashcards
What are the four main parts inside a spectrophotometer?
Lamps
Monochromator
Optics
Detector
The bigger the path length, the bigger the absorbance. True or false?
True
Why can’t normal glass be used for cuvette?
Because it has an absorbance of its own
Absorbance of the sample should be >1.5 otherwise dilution is recommended. True or false?
False <1.5
Why are samples with an absorbance of >1.5 diluted?
Because molecules can pack together and cause deviations
Cuvettes should be handled from the clear side, not frosted side to prevent transfer of proteins from the hand. True or false?
False - hold from the frosted side
What is derivative spectrophotometry and how does it differ from normal?
The normal plot of absorbance against wavelength is zero order.
In derivative spectrophotometry, the absorbance is differentiated with respect to wavelength and as a result, sharp waves become amplified
Derivates help to identify peaks, they do not increase data. True or false?
True
What is jablonski’s diagram?
An energy diagram that describes the process of photon emission
What is the only way in which fluorescence can be generated?
Electrons going from S1 to S0
Can electrons go from S2 to S0 directly?
No, have to go to S1 before S0
Phosphorescence is produced when electrons pass from S1 to ___
T1
The return of an electron from excited singlet state to ground state requires change in spin orientation. True or false?
False - doesn’t require
Phosphorescence has a longer lifetime than fluorescence. True or false?
True
Intersystem crossing requires spin orientation to change. True or false?
True
What is an internal conversion?
Radiationless transition, but vibrational levels need to match
What is intersystem crossing?
Molecules relax via a non-radiative transition to the T1. Requires spin orientation to change
Phosphorescence takes place when electrons return from a triplet excited state to ground state. True or false?
True
Why does phosphorescence occur at longer wavelengths than fluorescence?
Because the energy difference between S0 and T1 is lower
A chromophore that can emit fluorescence is called a _____
fluorophore
Describe the fluorescence process
Absorption of light leads to excitation to a higher vibrational state (either within S0 or to S1 or S2
Vibrational relaxation takes place, till the lowest vibrational state in S1 is achieved - the molecule may undergo conformational change to achieve this
Molecule relaxes from the lowest vibrational energy level of the excited state to one of the vibrational energy levels in S0 = fluorescence
What is stokes shift?
The difference in wavelength between absorption and emission
Why is the wavelength of light emitted higher than that of light absorbed?
Because so of the absorbed energy is lost due to process that happen in the excited state lifetime e.g. vibrational relaxation etc.
The excited state of electron lifetime is short. True or false?
True
What is on the x and y axis for an emission spectrum?
Fluorescence intensity on y
wavelength on x
Fluorescence is less sensitive than UV-visible light for detection of analytes. True or false?
False - more sensitive
Why is fluorescence more sensitive to UV-visible light for detection of analytes?
Because it can be repeated more than once - some molecules can be excited repeatedly to produce more photons
Emission is at a higher wavelength than absorption so background signal can be quite low at detection wavelength
Instruments that measure fluorescence are called?
Spectrofluorometers
What is a key difference in the instrumentation between spectrophotometers and spectrofluorometers?
Spectrofluorometers have two monochromators
The cell holder in fluorescence needs to be clear on all four sides. True or false?
True
What are the advantages of using fluorescence?
Higher selectivity than UV-vis
Inexpensive
High sensitivity
Can be applied in many cases without a separation step
What are the limitations of using fluorescence?
Not all drugs are fluorescent
Changes in conditions can affect fluorescence properties
Use of standards normally required in pharmaceutical analysis
What are the main factors that affect fluorescence intensity?
Inner filter effect pH Temp Viscosity Presence of oxygen Photobleaching
How does the inner filter effect affect fluorescence?
At high drug concentration, fluorescence intensity reaches a plateau. At further increase in concentration, intensity decreases because of inner-filter effects
Barbituates only fluoresce in the di-anionic form. True or false?
True
Phenol fluoresces at pH __ anion
7
How do viscosity and temperature have an effect on fluorescence intensity?
They have opposite effects - as molecules move more freely (low viscosity, high temp) more collisions take place and so they lose more energy leading to lower fluorescence
A high oxygen concentration quenches fluorescence through collisions. True or false?
True
Structural rigidity increases fluorescence. True or false?
True
Electron withdrawing groups lower fluorescence, while electron donating groups increase fluorescence. True or false?
True
What are the analytical problems that digoxin and digitoxin have?
They have weak absorbance at 220nm
They are potent drugs so only a small quantity is available
The excipients of the tablet also absorb at 220nm
The dehydration of the steroid molecule in digoxin and digitoxin with strong acids or oxidising agents generates analytically useful fluorphore form. True or false?
True
