Week 5 - Spectroscopy Flashcards
SPECTROSCOPY
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.”
What different about different spectroscopy methods?
different light frequency—gives a different picture -> the spectrum
What does a spectrum measure?
interaction of light with a sample which can influence the sample and/or the light
Spectrum method
method involves
- excitation
- light - EM wave
- other excitation sources - detection
- characterise light after sample
- characterises change in sample
absorption
ground state to excited state
relaxation
excited state to ground state
Internal conversion
non-radiative relaxation through vibrational states
Emission
exciting induces emission of light from the sample - usually of different frequencies
emits:
- fluorescence
- phosphorescence
- raman scattering
fluorenscence
emission from excited electronic singlet states
phosphorescence
emission from excited electronic triplet states
raman scattering
light scattering involving vibrational transition
ABSORPTION PROCESS
Transfer of light energy to
molecule
What happens next depends
on the characteristics of the
molecule
Photon
particles which transmit light
What event leads to the increase in energy level?
photon is absorbed by an atom or molecule
transitioned from lower energy level to higher
this transition depends on photon energy
What happens when sample absorbs electromagnetic radiation?
number of photons that pass through will decrease
molecule absorb photon
less light measured
What do we measure?
how much light we put in and how much that come out
What can we use the beer lambert law for?
to measure concentration
How is the concentration determined from the beer-lambert law?
through UV-viable spectroscopy
What is the primary detection from beer-lambert law?
light intensity is exponential attenuation with
- absorber molecule conc
- sample cross section - optical path length
What does polarisation give an idea of?
specific isomer
make sure only one stereoisomer
What do all crystals show?
anisotropy
What does anisotropy in crystals mean?
means that all certain or physical properties are different directions
- they are directional
What are examples of anisotropy?
- hardness, cleavbility
- elasticity, expansion properties
- electric/thermal conductivity
- electric polarzagbility - magnatism
How do we understand structure of crystal?
we can shine a light on the plane though to understand how the structure or crystal is
How do we measure absorption?
measure changes in light intensity as it passes through a sample
What is the excitation filter?
the absorbance we set at which filters what is coming through
What are the parts of a spectophotometer?
objective
excitation filter
dichromate mirror
emission filter
detector
light source
What are sorte peaks?
sharp intense peaks
What wavelength is used to detect the purity of DNA?
280nm
What wavelength is used to detect the quantify of DNA?
260nm for detection level
this also gives the concentration
What ratio are we looking at when purifying DNA?
1:8
What wavelengths do we look at to check the purity of a DNA sample?
260-280
Photoluminescence
is light emission from any form
of matter after the absorption of photons
Luminescence
emission of light – no heat
What are the types of emissions in emission spectroscopy?
Luminescence – emission of light – no heat
- Phosphorescence
- Fluorescence
- Chemiluminescence
What is the process of fluorescence?
Emission of a photon from the singlet excited state to the ground state.
Fluorescence decays rapidly once the source of excitation is removed
When does fluorescence stop?
stops when there is no energy
Fluorescence - Relaxation of excited state
- Collisions with other species in the
sample - Photochemical reactions
- Emission of photons
Quantum Yield of Fluorescence
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.
What are the two uses of fluorescence spectroscopy?
- 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. - Quantitative analysis
- At low concentrations, the fluorescence intensity of the solution is
directly proportional to the concentration of the fluorescent
substance:
USES OF FLUORESCENCE SPECTROSCOPY -
EXAMPLES
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
ADVANTAGES OF FLUORESCENCE SPECTROSCOPY
Fluorescence analysis
- 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.
What is FLUORESCENCE ANALYSIS suitable for?
suitable for analytes that can be dissolved in solvents like water, ethanol
and hexane
What do the analytes need to do in fluorescence analysis?
- analytes need to absorb UV or visible light
- analytes need to emit visible or near infra red radiation
What does fluorescence analysis measure?
quantitative measurements of a single analyte in solution
(Or more than one analytes in solution provided they do not interfere with each other.)
What don’t fluorescence analysis do?
- 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
What is excitation spectrum is obtained by?
monitoring emission at a fixed wavelength,
variable excitation wavelength
Emission spectrum
Fixed wavelength to excite sample
Monitor emitted wavelength intensity
What do molecules show on emission spectrum?
Molecules have single excitation spectrum
but two emission spectra – fluorescence,
phosphorescence
What is quenching?
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
What is quenching the bases of?
Förster resonance energy transfer (FRET) assays
Förster resonance energy transfer (FRET) assays
energy from excited molecular fluorphore (donor) is transferred to another fluorophore (acceptor)
What is the basic principle of FRET?
donor that donates and returns to ground state
What is a key thing about FRET?
- 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
FRET Key information
- 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
FRET - What happens if acceptor is fluorophores?
transferred energy emitted as fluorescence
FRET - What happens if acceptor is not fluorophores?
energy lost as heat and not light
FRAP
Fluorescence Recovery After
Photobleaching
What is FRAP?
a method of determining the kinetics of
diffusion in living cells (usually) using
fluorescence microscopy
FRAP - Steps
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
What does fluorescence measure?
determin kinetics
bleach out using dye
measure time it takes for it to get back to normal
= gives idea about diffusion
bioluminescence: FIREFLY FLASHES
In bioluminescence, a photon is released
after excitation in a biochemical
reaction, e.g. luciferase
bioluminescence - how is light produced?
luciferyl adenylate is converted to oxyluciferin using oxygen and the enzyme luciferase
Bioluminescence - what enzyme is used?
luciferase
What is LUCIFERASE ASSAYS used to determine?
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
Chemiluminescence
a photon is released after
excitation in a chemical reaction, e.g. luminol
What is luminol used to detect?
blood
What does laminal need to be activated?
needs to be oxidised to activate using hydrogen peroxides in water
catalyst required
What do you look at in luciferase essays?
looking at effects of protein on gene transcription
What does adding luciferase as part of promoter region do?
create moderated protein production and can look at the light signals that is produced from it
What is light directly proportional to in luciferase assay?
amount of biologically active chemical in sample
Process of lumionol activation
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)
What contains ROOR?
Organic peroxides contain peroxide
functional group ROOR
What is the NIR- absorption spectroscopy wavelength of interest?
- 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
NIR- ABSORPTION SPECTROSCOPY USES
- look at water hardness
- fermentation processes
What happens if the sample is turbid?
light propagation within the sample is no longer rectilinear and the transmitted light is partly missing the detector of the spectrometer
