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Analytical and Clinical > Week 5 - Spectroscopy > Flashcards

Week 5 - Spectroscopy Flashcards

1
Q

SPECTROSCOPY

A

Studying the properties of matter through its interaction with different
frequency components of the electromagnetic spectrum.

With light – interaction not with the matter but the “ghost.”

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2
Q

What different about different spectroscopy methods?

A

different light frequency—gives a different picture -> the spectrum

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3
Q

What does a spectrum measure?

A

interaction of light with a sample which can influence the sample and/or the light

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4
Q

Spectrum method

A

method involves

  1. excitation
    - light - EM wave
    - other excitation sources
  2. detection
    - characterise light after sample
    - characterises change in sample
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5
Q

absorption

A

ground state to excited state

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6
Q

relaxation

A

excited state to ground state

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7
Q

Internal conversion

A

non-radiative relaxation through vibrational states

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8
Q

Emission

A

exciting induces emission of light from the sample - usually of different frequencies

emits:

  • fluorescence
  • phosphorescence
  • raman scattering
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9
Q

fluorenscence

A

emission from excited electronic singlet states

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10
Q

phosphorescence

A

emission from excited electronic triplet states

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11
Q

raman scattering

A

light scattering involving vibrational transition

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12
Q

ABSORPTION PROCESS

A

Transfer of light energy to
molecule

What happens next depends
on the characteristics of the
molecule

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13
Q

Photon

A

particles which transmit light

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14
Q

What event leads to the increase in energy level?

A

photon is absorbed by an atom or molecule

transitioned from lower energy level to higher

this transition depends on photon energy

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15
Q

What happens when sample absorbs electromagnetic radiation?

A

number of photons that pass through will decrease

molecule absorb photon

less light measured

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16
Q

What do we measure?

A

how much light we put in and how much that come out

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17
Q

What can we use the beer lambert law for?

A

to measure concentration

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18
Q

How is the concentration determined from the beer-lambert law?

A

through UV-viable spectroscopy

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19
Q

What is the primary detection from beer-lambert law?

A

light intensity is exponential attenuation with

  1. absorber molecule conc
  2. sample cross section - optical path length
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20
Q

What does polarisation give an idea of?

A

specific isomer

make sure only one stereoisomer

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21
Q

What do all crystals show?

A

anisotropy

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22
Q

What does anisotropy in crystals mean?

A

means that all certain or physical properties are different directions
- they are directional

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23
Q

What are examples of anisotropy?

A
  • hardness, cleavbility
  • elasticity, expansion properties
  • electric/thermal conductivity
  • electric polarzagbility - magnatism
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24
Q

How do we understand structure of crystal?

A

we can shine a light on the plane though to understand how the structure or crystal is

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25
Q

How do we measure absorption?

A

measure changes in light intensity as it passes through a sample

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26
Q

What is the excitation filter?

A

the absorbance we set at which filters what is coming through

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27
Q

What are the parts of a spectophotometer?

A

objective

excitation filter

dichromate mirror

emission filter

detector

light source

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28
Q

What are sorte peaks?

A

sharp intense peaks

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29
Q

What wavelength is used to detect the purity of DNA?

A

280nm

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30
Q

What wavelength is used to detect the quantify of DNA?

A

260nm for detection level
this also gives the concentration

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31
Q

What ratio are we looking at when purifying DNA?

A

1:8

32
Q

What wavelengths do we look at to check the purity of a DNA sample?

A

260-280

33
Q

Photoluminescence

A

is light emission from any form
of matter after the absorption of photons

34
Q

Luminescence

A

emission of light – no heat

35
Q

What are the types of emissions in emission spectroscopy?

A

Luminescence – emission of light – no heat
- Phosphorescence
- Fluorescence
- Chemiluminescence

36
Q

What is the process of fluorescence?

A

Emission of a photon from the singlet excited state to the ground state.

Fluorescence decays rapidly once the source of excitation is removed

37
Q

When does fluorescence stop?

A

stops when there is no energy

38
Q

Fluorescence - Relaxation of excited state

A
  • Collisions with other species in the
    sample
  • Photochemical reactions
  • Emission of photons
39
Q

Quantum Yield of Fluorescence

A

fraction of excited state molecules
returning to the ground state by
fluorescence range from 1, when every
molecule in an excited state
undergoes fluorescence, to 0
when fluorescence does not occur.

40
Q

What are the two uses of fluorescence spectroscopy?

A
  1. Qualitative analysis
    - shapes and peak positions of the excitation spectra and emission
    spectra of fluorescent substances can be compared with the spectra of standard solutions to achieve the purpose of qualitative
    analysis.
  2. Quantitative analysis
    - At low concentrations, the fluorescence intensity of the solution is
    directly proportional to the concentration of the fluorescent
    substance:
41
Q

USES OF FLUORESCENCE SPECTROSCOPY -
EXAMPLES

A

Quantification - DNA, antibodies, antigens

Drug analysis, pharmacokinetics, efficacy
analysis – e.g. quinine, antimicrobial drugs

Food – detecting minerals, metallic elements, aas, vitamins, fungal contamination etc

Environmental analysis – water, soils

Currently not for treatment or diagnosis

42
Q

ADVANTAGES OF FLUORESCENCE SPECTROSCOPY
Fluorescence analysis

A
  • qualitatively and quantitatively analyse substances
    based on characteristics and intensity of
    fluorescence produced
  • widely used to characterise physical and chemical
    properties of the system and its changes – e.g. conformation and properties of biological macromolecules.
43
Q

What is FLUORESCENCE ANALYSIS suitable for?

A

suitable for analytes that can be dissolved in solvents like water, ethanol
and hexane

44
Q

What do the analytes need to do in fluorescence analysis?

A
  • analytes need to absorb UV or visible light
  • analytes need to emit visible or near infra red radiation
45
Q

What does fluorescence analysis measure?

A

quantitative measurements of a single analyte in solution

(Or more than one analytes in solution provided they do not interfere with each other.)

46
Q

What don’t fluorescence analysis do?

A
  • Analytes that have a photochemical reaction at (or above) the wavelength
    range of interest
  • Intransparent, not clear or colloidal samples
  • Compounds that do not show fluorescence
47
Q

What is excitation spectrum is obtained by?

A

monitoring emission at a fixed wavelength,
variable excitation wavelength

48
Q

Emission spectrum

A

Fixed wavelength to excite sample

Monitor emitted wavelength intensity

49
Q

What do molecules show on emission spectrum?

A

Molecules have single excitation spectrum
but two emission spectra – fluorescence,
phosphorescence

50
Q

What is quenching?

A

Quenching and dequenching is the basis for activatable optical contrast agents for molecular imaging.

Many dyes undergo self-quenching, which can decrease the brightness of protein-dye conjugates for fluorescence microscopy, or can be harnessed in sensors of proteolysis

51
Q

What is quenching the bases of?

A

Förster resonance energy transfer (FRET) assays

52
Q

Förster resonance energy transfer (FRET) assays

A

energy from excited molecular fluorphore (donor) is transferred to another fluorophore (acceptor)

53
Q

What is the basic principle of FRET?

A

donor that donates and returns to ground state

54
Q

What is a key thing about FRET?

A
  • need relativly small distance between donor and acceptor
  • once energy is transferred over = flourece
  • if it doesn’t fluorecese we lose that energy as heat
55
Q

FRET Key information

A
  • Energy level of donor –returns to
    ground state without own fluorescence
  • Limited distance between two
    molecules
  • Emission spectrum of donor overlaps
    absorption spectrum of acceptor
  • If acceptor is fluorophore – transferred
    energy emitted as fluorescence
  • If acceptor not fluorophores – energy
    lost as heat and not light
56
Q

FRET - What happens if acceptor is fluorophores?

A

transferred energy emitted as fluorescence

57
Q

FRET - What happens if acceptor is not fluorophores?

A

energy lost as heat and not light

58
Q

FRAP

A

Fluorescence Recovery After
Photobleaching

59
Q

What is FRAP?

A

a method of determining the kinetics of
diffusion in living cells (usually) using
fluorescence microscopy

60
Q

FRAP - Steps

A

A. The bilayer is uniformly labelled with a fluorescent tag

B. label is selectively photobleached by a small (~30micrometre) fast light pulse

C. The intensity within this bleached area is monitored as the bleached dye diffuses out and new dye diffuses in

D. Eventually uniform intensity is restored

61
Q

What does fluorescence measure?

A

determin kinetics

bleach out using dye

measure time it takes for it to get back to normal

= gives idea about diffusion

62
Q

bioluminescence: FIREFLY FLASHES

A

In bioluminescence, a photon is released
after excitation in a biochemical
reaction, e.g. luciferase

63
Q

bioluminescence - how is light produced?

A

luciferyl adenylate is converted to oxyluciferin using oxygen and the enzyme luciferase

64
Q

Bioluminescence - what enzyme is used?

A

luciferase

65
Q

What is LUCIFERASE ASSAYS used to determine?

A

To determine if a protein is able to activate (or suppress) transcription of a gene of interest, recombinant DNA technology to produce a construct in which the gene’s promoter is placed adjacent to a luciferase reporter gene

66
Q

Chemiluminescence

A

a photon is released after
excitation in a chemical reaction, e.g. luminol

67
Q

What is luminol used to detect?

A

blood

68
Q

What does laminal need to be activated?

A

needs to be oxidised to activate using hydrogen peroxides in water

catalyst required

69
Q

What do you look at in luciferase essays?

A

looking at effects of protein on gene transcription

70
Q

What does adding luciferase as part of promoter region do?

A

create moderated protein production and can look at the light signals that is produced from it

71
Q

What is light directly proportional to in luciferase assay?

A

amount of biologically active chemical in sample

72
Q

Process of lumionol activation

A

Luminol reacts with OH – dianion

Dianion reacts with O2 to produce
peroxide – unstable – made by loss of
N2, and change of electrons from
excited to ground state – emission of
photons (blue glow)

73
Q

What contains ROOR?

A

Organic peroxides contain peroxide
functional group ROOR

74
Q

What is the NIR- absorption spectroscopy wavelength of interest?

A
  • The interesting wavelength range for this type of spectroscopy is 800-2500 nm (invisible).

– The related wavenumber (1/λ) range is 12500 – 4000 cm-1

75
Q

NIR- ABSORPTION SPECTROSCOPY USES

A
  • look at water hardness
  • fermentation processes
76
Q

What happens if the sample is turbid?

A

light propagation within the sample is no longer rectilinear and the transmitted light is partly missing the detector of the spectrometer

Analytical and Clinical (11 decks)

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