Spectrophotometry Flashcards
True or false: wavelength is proportional to a given frequency
False its inversly proportional
v=c/lamda
How can light be absorbed?
Electrons in ground state are promoted to higher orbitals. Energy absorbed correspond to difference between orbitals
- Energy is then lost either vibrationally or radiatively
Why is organic molecules particularly useful for this?
Often have a conjugated double bond systems, so easy for delocalisation.
- Double bond systems have fairly low energies for absorption
- Since colour occurs with absorption, many organic conjugated compounds are coloured
What is the beer lambert law?
Conc of a substance is directly proportional to the amount of light absorbed. Expressed as A= Ecl
E- molar absorption coeff
Why is absorbance an ideal analysis?
- Cheap relating to use
- Measurement is quick and easy
- Readily integrated into automation
- Reproducible
Define spectrophotometry
the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.
Light passes through solution and detected by photodetector generating an electrical current proportional to intensity of light
Evaluate different light sources.
Tungsten
- Covers spectra well
- Higher intensity around red
- Cheap
Deuterium lamp
- use isotopic hydrogen
- Produce mainly UV light
- Expensive
- Short lifetime
Tungsten covers a greater range but only one section has a higher specificity
Properties of the light beam
Incident light beam is parallel and of a constant wavelength (monochromatic). Incident light beam is of the wavelength which gives the max absorption. Achieved using a prism (rotate) or grating to isolate proportion of spectra of white light from the bulb
Purpose of using a prism or diffraction grating
To isolate proportion of spectra of white light from the bulb
What purpose does holmium oxide serve to MS?
Has a complex absorption spectra with sharp, well defined peaks across the visible range making it a good calibrator
What are some different detectors?
Photomultiplier tube (very high sensitivity)
- exponentially amplify light signal
- cascade system of dynodes
- converts light to electron beam then to electric current
- relatively expensive
Diode array
- slightly different way to conventional spectrophotometres
- The array contains several light detectors
- Several wavelengths of light can be measured simoultaneously
What is a dual beam spectrophotometer?
Where incident beam is split by mirror
Allows correction for drift and power variation in the light source
- Beam is split to go through both sample and reference sample
State forms of spectrophotometry
- Reflectance e.g. bilirubin levels in neonates
- Scatter
- Fluorescence
- Luminometry
What is immunoturbidimetry?
- Antibodies bind to the analyte
- Form aggregates which scatter light
- Amount of scatter is proportional to amount of analyte
- Sensitivity can be increased by attaching antibodies to latex or polystyrene particles
What is nephelometry?
More sensitive assays because they measure light scattered at 90degree angle.
Require specialist equipment.
What is fluoresence?
Occurs when a molecule absorbs one wavelength and emits at a longer wl
- emitted light is always lost at a longer wavelength than excitation light
- Stokes shift: difference between max wavelength of excitation light and emitted fluoresence light
Where is fluorescence used?
Immunoassay detection systems - DELFIA
Polarisation methods - FPIA
FISH
PCR
What is a luminescence?
Generation of light from chemcial reaction
- extremely sensitive
Energy of photons is…….. to its frequency
Proportional E=hv h is planks constant 6.62x10power of27 v= frequency of light E=energy
What is E=hc/lambda based on?
The fact that wavelength is inversely proportional to frequency and frequency is directly proportional to energy
