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spectrometry and fluorometry - Coggle Diagram
spectrometry and fluorometry
the the higher the frequency of the electromagnetic radiation, the shorter the wave length
each element has a specfic emission spectrum that can be used as a
fingerprinting identification
of that element
the shorter the wavelength/the higher the frequency, the higher the energy
electromagnetic radiation can behave as waves so it can be reflected or refracted. They can also behave like small particle-like substances-photons
Properties
travel through space(vacuum), lose some of their energy when they come into contact with other particles and re-emit as photons of lower energy
Before excitation, the electrons are in their lowest electronic energy state-ground state(E0)
When atoms interact with light/radiation, the electrons absorb the energy and become excited to a higher energy state(E1)
electron does not stay in the excited state for long, it will release(re-emit) the energy in the form of another photon,
electron will return to the ground state
photon will have lower energy level as some energy is lost as heat
electrons having fixed energy levels will only absorb photons with corresponding energy level
chromophores and pigments
organic molecules that contains pi electrons are able to absorb UV-Vis radiation
many naturally occurring chromophore and pigment molceules also have conjugated bonds or pi delocalisation-contribute to their ability to absorb visible light
pi delocalisation-sharing of electrons among conjugated pi bonds
Fluorescence - The ability of a molecule to emit visible radiation. This requires excitation of the molecule by electromagnetic radiation from a separate source
beer-lambert law is only valid for low concentration of analyte
at high conc, individual particles of analyte no longer behave independently of one another
the value of ε changed
the graph is no longer linear
fluorochromes/fluorophores are molecules capable of emitting visible light when excited by a higher energy radiation
process whereby fluorochromes are excited by UV radiation and emit visible light as they return to their ground state is known as fluorescence
excitation-energy supplied by an external energy source and electron moves to an excited state
excitation state exists for a finite time, energy is dissipated, yielding a lower energy state
emission-photon is emitted and electron returns to ground state
the process is cyclical unless the fluorochrome is irreversibly destroyed in the excitation state
fluorochrome has its absorption and emission spectra
stokes shift-diff between maxima of excitation and emission spectra-should be large enough(25 to 30nm) to prevent interference in fluorescence microscopy
application of fluorochromes
imaging
certain fluorochromes bind to specific organelles(rhodamine 123 to mitochondria and acridine orange to nucleoli
membrane probes-fluorescent lipids have been developed to study vesicle transport
physiological indicators and tracers-some fluorochromes change its fluorescence spectra at diff pH or when bound to specific ions