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Analytical methods > Spectrophotometry > Flashcards

Spectrophotometry Flashcards

1
Q

True or false: wavelength is proportional to a given frequency

A

False its inversly proportional

v=c/lamda

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2
Q

How can light be absorbed?

A

Electrons in ground state are promoted to higher orbitals. Energy absorbed correspond to difference between orbitals
- Energy is then lost either vibrationally or radiatively

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3
Q

Why is organic molecules particularly useful for this?

A

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
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4
Q

What is the beer lambert law?

A

Conc of a substance is directly proportional to the amount of light absorbed. Expressed as A= Ecl
E- molar absorption coeff

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5
Q

Why is absorbance an ideal analysis?

A
  • Cheap relating to use
  • Measurement is quick and easy
  • Readily integrated into automation
  • Reproducible
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6
Q

Define spectrophotometry

A

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

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7
Q

Evaluate different light sources.

A

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

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8
Q

Properties of the light beam

A

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

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9
Q

Purpose of using a prism or diffraction grating

A

To isolate proportion of spectra of white light from the bulb

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10
Q

What purpose does holmium oxide serve to MS?

A

Has a complex absorption spectra with sharp, well defined peaks across the visible range making it a good calibrator

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11
Q

What are some different detectors?

A

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
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12
Q

What is a dual beam spectrophotometer?

A

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

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13
Q

State forms of spectrophotometry

A
  • Reflectance e.g. bilirubin levels in neonates
  • Scatter
  • Fluorescence
  • Luminometry
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14
Q

What is immunoturbidimetry?

A
  • 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
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15
Q

What is nephelometry?

A

More sensitive assays because they measure light scattered at 90degree angle.

Require specialist equipment.

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16
Q

What is fluoresence?

A

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
17
Q

Where is fluorescence used?

A

Immunoassay detection systems - DELFIA

Polarisation methods - FPIA

FISH

PCR

18
Q

What is a luminescence?

A

Generation of light from chemcial reaction

- extremely sensitive

19
Q

Energy of photons is…….. to its frequency

A 
Proportional 
E=hv
h is planks constant 6.62x10power of27
v= frequency of light 
E=energy
20
Q

What is E=hc/lambda based on?

A

The fact that wavelength is inversely proportional to frequency and frequency is directly proportional to energy

Analytical methods (15 decks)

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