What are the key components of a spectrophotometer?
- Source that generates a broad band of electromagnetic radiation.
- Dispersion device selects broadband radiation.
- Sample Area
- 1+ detectors to measure intensity of radiation
What does it mean if an instrument is double beam?
Light will pass through mirror/lense and split into two beams, giving both a reference and sample. This stops light source drift problem that occurs in single beam
What are the two kinds of light sources in analytical spectrophotometry?
A Tungsten lamp (350-2000nm, suitable for colourimetry) Deuterium lamp (200-370nm, good for UV)
What are the two types of monochromator and what must they contain?
- Prisms
- Diffraction gratings
They must all contain an entrance slit, a collimating mirror (or lens), a dispersing device, a focusing mirror (or lens) and an exit slit. They work by selecting a particular wavelength via Prisms or diffraction gratings.
What is diffraction grating?
It is where radiation enters and the beam is collimated and strikes a dispersing element at an angle, the beam is split into component λ by grating.
What does a detector do and what are the two main types?
Converts a light signal- electrical signal. Photomultiplier tube and photodiode array exist.
Explain how a photomultiplier tube works
Electrons given off, attracted to a dynode, give off more electrodes, reach anode. Amplifies signal.
Explain how a photodiode array works
Liquid passes through, no time to stop, diode array constantly measuring.
What may cause deviations from Beer’s law?
Instrumental: Non-linearity may be caused by stray light in the instrument at high absorbances, causing an underreported value of absorbance.
Chemical: pH- some chromophores shift wavelength with changed pH, this can be overcome using a buffer
Concentration: Above 10mM the close distance between absorbing molecules can alter ability to absorb a given λ
What is a bathochromic (red) shift?
A shift in λ max towards longer wavelength. Usually due to action of an auxochrome (a functional group attached to the chromophore which doesnt absorb light energy itself but influences the wavelength absorbed by chromophore).Auxochromes possess a lone pair of electrons which interact with the Pi electrons of chromophore. Usually associated with decreased light absorption (hypo chromic)
What is a hypsochromic (blue) effect?
A shift in λ max to a shorter wavelength. Occurs when compounds with a BASIC auxochrome ionise and lone pairs can’t interact with electrons of chromophore. Usually associated with increases in light absorption (hyper chromic)
What effect does increasing light source intensity have on absorption?
No effect
What is the difference between a comparative and absolute assay method?
Comparative needs to know concentration.
Absolute must use Beer’s law.
What is fluorimetry?
Analytical technique that relies on the EMISSION of electromagnetic energy by molecules. The light emitted by the sample is always of longer wavelength (lower energy) than the light absorbed by the molecule.
What is internal conversion?
A non-radiative transition between states of the same multiplicity. No light is given off in this process. It is possible to get IC back to ground state but the energy gap is much bigger so less efficient. The loss of energy here accounts for Stokes shift in wavelength from excitation to emission.
What is Inter-system crossing?
ISC is when the spin of an excitated electron is reversed (singlet to triplet transition).
Explain the mirror image rule
The differences between vibrational levels are similar in the ground and excited states so that the fluorescence spectrum resembles the first absorption band.
What is Stokes shift?
The gap between the maximum of the first absorption band and the maximum of fluorescence.
What is a spectrofluorimeter made up of?
A high energy light source (Xenon arc lamp) 2 monochromators (excitation and emission) Detector aligned 90 degrees to lamp to minimise high energy light reaching detector that has not passed through sample. Detector usually PMT as signals usually weak.
What is special about the dual monochromator spectrofluorimeter?
It has 2 grating monochromator so can give operator choice of wavelength for both excitation and emission. Able to scan for λ max of both.
What are the advantages of fluorimetric analysis?
More specific than UV.
Lower detection limit than UV/Vis.
Easier to measure a small signal than the difference between two large signals.
Fluorescence intensity can be increased easily.
What factors affect fluorescence?
Source intensity Fluorescence efficiency Concentration Pathlength Quenching Self absorption
What is quenching?
A reduction in the intensity of light emitted during fluorescence. Often caused by other things in the sample.
Two types= self of chemical.
Explain the mechanism of fluorescence
Excitation- vibrational relaxation (IC), drops down to lowest excited state- fluorescence= dropping down
Why is a high power light source used in fluorescence but not in absorption?
Maximises intensity, more light in= more fluorescence out. Doesn’t work in absorption
How does atomic emission work?
Uses energy from the flame to excite electrons into excited state and then relaxes back to ground state, giving off a photon of light.
What energy is required in order to cause an electron to be ionised?
More than 5.2 eV
What does spectral line width describe?
The narrow spread of wavelengths over which absorption and emission is observed for a given electronic transition
What is ICP-OES?
Used in atomic emission
An ICP torch is a device that produces a plasma, very efficient atomisation source. It exposes the sample to high temperature plasma and converts a very large proportion of atoms to an excited state. Has a coolant gas to stop from melting. Uses argon gas.
What are the advantages of Inductively coupled plasma (ICP)?
Better and safer than flame, it can be left alone whereas flame needs continuously watched. Offers simultaneous analysis. High temperature of plasma gives large choice of emission lines. Good detection limits. Good precision.
What is the technique of ICP?
Ar gas enters, RF energy applied, sparks cause some Ar to ionise and form free electrons which are then accelerated by RF fields, cause further ionisation and plasma forms. Flow from nebuliser carries sample through.
What interferences may occur in ICP?
Spectral- overlapping. Must select different non-overlapping lines.
Chemical- low volatility compounds not readily atomised
Sulphate and phosphate form involatile salts with metals and reduce sample reading
Remove by adding lanthanum chloride which precipitates them out and replaces them with chloride anions
Matrix effects- may alter density, viscosity which alter rate of aspiration.
What is a ‘standard addition’ used in ICP?
Used to eliminate matrix effects (interference due to excipients in sample). Increasing volumes of standard solution added to sample. Increases accuracy and precision of assay. Works to try and determine interferences
Explain how a flame photometer works in atomic emission
Needs to be liquid, nebuliser makes fine droplets, then flame.
What are the differences between atomic emission and atomic absorption?
In AE a flame is used to excite electrons from ground state to excited state (Boltzmann Law)
In AA flame is only used to atomise the sample. Absorption is carried out using a hollow cathode lamp
How does atomic absorption work?
It uses a light source of specific wavelength to cause the electron to be promoted to the excited state. The flame is used to atomise the sample. The amount of light absorbed is measured.
What is a hollow cathode lamp? (Used in atomic absorption)
Its cathode is made of the same metal as sample and emits light of exactly the same wavelength as absorbed by the sample.If element you want to measure changes, need to change lamp. Very specific to that element.
How does a hollow cathode lamp work?
Highly energetic electrons emitted by cathode, Ar ionised by collision, accelerated to cathode causing sputtering (process of producing gas atoms directly from a solid metal) Metal atoms are excited by collisions with electrons and ions which then emit the characteristic atomic emission lines. Limitation= not good for volatile elements.
Explain deuterium lamp background correction.
Deuterium lamp emits broad spectrum of light. Light passing the sample in the flame is alternated between the HCL and the deuterium lamp. Broad spectrum not affected by narrow absorbance of atom = background. Whilst HCL is affected by absorbance by atoms = signal. Able to subtract the two to leave just the AA signal.
Why is atomic absorption reproducible?
It does not rely on temperature of flame or flow rate of gas.
What are the disadvantages of atomic absorption?
Only determines one metal atom at a time. Have to change lamps for multi-component assays.
Doesn’t determine non-metals. Expensive.
How can atomic absorption instruments be adapted for atomic emission?
Remove light source
Rotate burner head (reduces self-absorption)
Reduce monochromator to lowest setting to give a lower chance of overlapping
How does Graphite furnace AA differ from FAAS?
You remove the flame and replace with a graphite furnace, its safer and has no need for a nebuliser. Is able to analyse solids directly, not needing to make them into a solution. Electrical heating is used to volatiles and atomise sample.
How does the L’vov platform work?
This is the concept that the platform is not heated directly by an electric current in graphite furnace, but by radiation and convection from furnace walls. This delays atomisation until the gas is heated up sufficiently to prevent condensation.
What are the advantages of graphite furnace AA?
Sample added directly so no need for nebuliser. Detection limits better than flame. Atoms present in atom cell for a relatively long time. Only small amounts of sample required. Potential for direct analysis of solids. No flammable gases used, safer and can be left unattended.
