Analytical Spec

Question 1

What are the key components of a spectrophotometer?

Answer 1

1. Source that generates a broad band of electromagnetic radiation.
2. Dispersion device selects broadband radiation.
3. Sample Area
4. 1+ detectors to measure intensity of radiation

Question 2

What does it mean if an instrument is double beam?

Answer 2

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

Question 3

What are the two kinds of light sources in analytical spectrophotometry?

Answer 3

A Tungsten lamp (350-2000nm, suitable for colourimetry)
Deuterium lamp (200-370nm, good for UV)

Question 4

What are the two types of monochromator and what must they contain?

Answer 4

1. Prisms
2. 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.

Question 5

What is diffraction grating?

Answer 5

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.

Question 6

What does a detector do and what are the two main types?

Answer 6

Converts a light signal- electrical signal. Photomultiplier tube and photodiode array exist.

Question 7

Explain how a photomultiplier tube works

Answer 7

Electrons given off, attracted to a dynode, give off more electrodes, reach anode. Amplifies signal.

Question 8

Explain how a photodiode array works

Answer 8

Liquid passes through, no time to stop, diode array constantly measuring.

Question 9

What may cause deviations from Beer’s law?

Answer 9

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

Question 10

What is a bathochromic (red) shift?

Answer 10

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)

Question 11

What is a hypsochromic (blue) effect?

Answer 11

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)

Question 12

What effect does increasing light source intensity have on absorption?

Answer 12

No effect

Question 13

What is the difference between a comparative and absolute assay method?

Answer 13

Comparative needs to know concentration.

Absolute must use Beer’s law.

Question 14

What is fluorimetry?

Answer 14

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.

Question 15

What is internal conversion?

Answer 15

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.

Question 16

What is Inter-system crossing?

Answer 16

ISC is when the spin of an excitated electron is reversed (singlet to triplet transition).

Question 17

Explain the mirror image rule

Answer 17

The differences between vibrational levels are similar in the ground and excited states so that the fluorescence spectrum resembles the first absorption band.

Question 18

What is Stokes shift?

Answer 18

The gap between the maximum of the first absorption band and the maximum of fluorescence.

Question 19

What is a spectrofluorimeter made up of?

Answer 19

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.

Question 20

What is special about the dual monochromator spectrofluorimeter?

Answer 20

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.

Question 21

What are the advantages of fluorimetric analysis?

Answer 21

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.

Question 22

What factors affect fluorescence?

Answer 22

Source intensity
Fluorescence efficiency
Concentration
Pathlength
Quenching
Self absorption

Question 23

What is quenching?

Answer 23

A reduction in the intensity of light emitted during fluorescence. Often caused by other things in the sample.
Two types= self of chemical.

Question 24

Explain the mechanism of fluorescence

Answer 24

Excitation- vibrational relaxation (IC), drops down to lowest excited state- fluorescence= dropping down

Question 25

Why is a high power light source used in fluorescence but not in absorption?

Answer 25

Maximises intensity, more light in= more fluorescence out. Doesn’t work in absorption

Question 26

How does atomic emission work?

Answer 26

Uses energy from the flame to excite electrons into excited state and then relaxes back to ground state, giving off a photon of light.

Question 27

What energy is required in order to cause an electron to be ionised?

Answer 27

More than 5.2 eV

Question 28

What does spectral line width describe?

Answer 28

The narrow spread of wavelengths over which absorption and emission is observed for a given electronic transition

Question 29

What is ICP-OES? | Used in atomic emission

Answer 29

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.

Question 30

What are the advantages of Inductively coupled plasma (ICP)?

Answer 30

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.

Question 31

What is the technique of ICP?

Answer 31

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.

Question 32

What interferences may occur in ICP?

Answer 32

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.

Question 33

What is a 'standard addition' used in ICP?

Answer 33

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

Question 34

Explain how a flame photometer works in atomic emission

Answer 34

Needs to be liquid, nebuliser makes fine droplets, then flame.

Question 35

What are the differences between atomic emission and atomic absorption?

Answer 35

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

Question 36

How does atomic absorption work?

Answer 36

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.

Question 37

What is a hollow cathode lamp? (Used in atomic absorption)

Answer 37

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.

Question 38

How does a hollow cathode lamp work?

Answer 38

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.

Question 39

Explain deuterium lamp background correction.

Answer 39

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.

Question 40

Why is atomic absorption reproducible?

Answer 40

It does not rely on temperature of flame or flow rate of gas.

Question 41

What are the disadvantages of atomic absorption?

Answer 41

Only determines one metal atom at a time. Have to change lamps for multi-component assays. Doesn't determine non-metals. Expensive.

Question 42

How can atomic absorption instruments be adapted for atomic emission?

Answer 42

Remove light source Rotate burner head (reduces self-absorption) Reduce monochromator to lowest setting to give a lower chance of overlapping

Question 43

How does Graphite furnace AA differ from FAAS?

Answer 43

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.

Question 44

How does the L'vov platform work?

Answer 44

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.

Question 45

What are the advantages of graphite furnace AA?

Answer 45

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.