MAAC: Analytical Spectrophometry 3 Flashcards
What is fluroescence used for in drug anaylsis
•Wide selection of applications for fluorescence in drug analysis
–Determination of fluorescent drugs in low-dose formulations (in the presence of non-fluorescent excipients)
–Carry out limit tests where impurity is fluorescent or can be rendered fluorescent
–For studying the binding of drugs to components in complex formulations
–Used in bioanalysis for measuring small amounts of drugs and studying drug-protein binding
What is the basic mechanism behind fluorimetry.
What is the difference in the wavelength of light absorbed and emitted? What is this called?
An analytical technique reli-ing on the emission of EM energy by molecules:
- Sample absorbs energy in UV region
- Sample emitts energy in Visiable light region
The wavelength emitted is ALWAYS of longer wavelength (lower E) than that of the absorbed. This (difference in wavelength) is called Stoke’s Law
Describe the basics of how fluorescence works in terms of transitions to different levels
- Electron absorbs energy and moves from ground state to excited vibrational state (A)
- The electron cools and internal conversion occurs - this is when the electron falls to the lowest energy level of the excited state by vibrational relaxation
From here 2 processes can occur
- The electron falls back down to ground state and fluroureces - gives off the excess energy in the form of a photon of light with a specific wavelength
OR
- The electron can under go intersystem crossing where it moves to a tripplet state. Here it falls to the lowest energy level in the excited tripplet state again (internal conversion) through vibrational relaxation. It then falls back to ground state in phosphorerescence
There are multiple types of excited states:
- Singlet states
- Tripplet states
What happens when to the electron when it is promote to each of these?
- Singlet states - Electron is paired and has an opposite spin to that of the ground state electron
- Tripplet states - Electron is unpaired and spin is parallel to ground state electron
The tripplet state is of lower energy than the singlet state. The tripplet state is also spectroscopically forbinden and less likely to occur.
During absorbance where do electrons transition?
Electrons absorb energy and can be promoted to any excited vibrational energy level
What is internal conversion and what happens during this process?
Internal conversion is a non-radiative transition between states of the same multiplicity.
As it is non-radiative no light is given out and it is lost/ transferred as heat to the solvent.
This loss in Energy between excitation and emission accounts for Stokes shift in wavelength.
In internal conversion the electron in the excited vibrational state undergoes vibrational relaxation to the lowest vibrational level of the final excited state (s1). This process occurs no matter which level the electon has been promoted too.
It is possible for the electron to return to ground state through internal conversion too, however the large difference in energy gap makes it unlikely to occur. The small energy gaps between excited states means IC takes place.
What is intersytem crossing?
Describe the movement of an electron from ISC to Gs
Inter-system crossing is when the spin of an excited e- is reversed. This occurs during a singlet – triplet transition.
It is also a non-radiative transition.
- Loss of energy from triplet state to ground state is spectroscopically forbidden
- Triplet state has long lifetime
- If this does occur radiatively then called phosphorescence
What is the relationship between wavelengths and transisitions?
What is the difference in wavelength emitted in Flucoeresences and phosphoresence?
Smaller difference between transitions = longer wavelength
Fluro - shorter
Phosp - longer
What can be said about the differences in energy levels in excited and Ground states?
What is stokes shift?
Absorped at 350nm and emitts at 380nm
- In general the differences between the vibrational levels are similar in the ground and excited states so that fluorescence spectrum resemble the first absorption band
- Mirror image rule
- The gap between the maximum of the first absorption band and the maximum of fluorescence is called the Stokes shift
- If absorbs at 380nm and emitts 350nm then stokes is 30nm
What components are required for a specttofluormeter?
Similar to spectrophotometer:
- High energy light source (Xenon arc lamp)
- 2x monochromators - one for emission and one for excitation
- Detector aligned at 90 degrees to lamp to minimise high energy reaching the detector that has not passed through the sample
- Usually photomultiplier used as detector and amplifier
What calculation is used to for quantative analysis?
Describe how a spectrofluorimeter works
- High energy Xenon arc lamp used
- Light passes through monochromator which converts polychormatic light to monochromatic ans selects desired wavelength
- Monochromatic light strikes sample and fluorescent light is given off at 360 degrees.
- The photomultipler detector is placed 90 degrees to the sample to avoid detecting any high energy light that has just passed straight through the sample
- Emission monochromator used to isolate emission wavelength. This means we have emission and excitation wavelength ans can specify these values and calculate stokes.
- PMT detects reading
- On the LHS there is a beam attenuator used to reduce light hitting detector so it doesn’t destory it. The light passes through this and into the reference PMT used to standaridse light intesnity across all wavelength
- What spectrofluorimeter is most versitile and why?
- Why is a PMT used?
- Why is a xenon light used?
- Most versatile is the dual monochromator spectrofluorimeter
- Two grating monochromators:
- Gives operator choice of wavelength for both excitation and emission
- Able to scan for λmax of both excitation and emission
- Fluroesence signals are generally weak therefore PMT can amplify
- Since fluorescence intensity is proportional to I0, a high power xenon lamp is used. Also covers all wavelengths in UV and Vis region
What is the advantage of flurimetric analysis compared to UV/ VIS spectroscopic analysis?
- •Fluorimetry is more specific than ordinary UV spectroscopy.
- Two monochromators + not all molecules with a chromophore fluoresce (cuts samples down).
- 2 samples with sample lamda max will still have diff emission wavelngth
- Two monochromators + not all molecules with a chromophore fluoresce (cuts samples down).
- •Fluorimetry has a detection limit approximately 100 times lower than UV/Visible absorption spectroscopy.
- Ideal for the analysis of very small amounts of potent drugs (e.g. contraceptive pill, 30 mg active steroid or drug metabolism studies).
- Intrinsically easier to measure a small signal (fluorescence intensity) than the difference between two large signals (I and I0) in absorbance
- Fluorescence intensity may be increased by increasing I0 (taking care to avoid photodecomposition)
What are factors influencing Fluorescence intensity?
What is self quencing?
- Seen at high concentrations (> 0.005%)
- Detection collects fluorescence emitted only from the central part of the exciting beam
- When concentration is large, a significant part of incident light is absorbed before reaching the central part of the cuvette
What is chemical quenching?
