Fluorescence Spectrophotometry Flashcards
method used to investigate molecular and atomic interactions by examining the fluorescence emitted from a sample
fluorimetry and spectrofluorimetry
type of luminescence caused by photons exciting a molecule, raising it to an electrnic excited state
fluorescence
method used to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution
Spectrophotometry
- process begins with the absorption of photon by a molecule, called the fluorophore, which results in the promotion of an electron to a higher energy state
- aborbed energy excites the molecule from its ground state to an excited state
excitation
absorbs photons and emits photons of lower energy in return
fluorophore
as excited molecule returns to the ground state, it emits a photon of lower energy than the absorbed photon
emission
- illustrates the transition of electronic states during fluorescence
- shows the energy levels and the absorbance spectrum of a typical fluorescent molecule
Jablonski Diagram
- refers to the average time or duration a fluorophore spends in the excited state before releasing a photon and returning to its ground state
- can vary from picoseconds to hundreds of nanoseconds depending on the fluorophore
fluorescence lifetime (FLT)
General Componenets of fluorescence spectrophotometry
- light source
- monochromator
- sample holder
- detector
- data output system
can emit radiation in the ultraviolet, visible, and near-infrared wavelengths
light source
selects a specific wavelength of light from the broad spectrum emitted by the light source
monochromator
Parts of the monochromator
- entrace slit
- collimating lens
- diffraction grating or prism
- exit slit
controls the width of the incident light beam
entrance slit
converts divergent light rays into parallel rays
collimating lens
selects a narrow range of wavelengths for excitation
diffraction grating or prism
determines the bandwidth of the selected excitation wavelength
exit slit
- where the sample to be analyzed is placed
- usually quartz cuvette or a microplate, designed to hold the sample securely and allow the excitation light to pass through it
sample holder
sample holders are usually what?
- quartz cuvette or
- microplate
measures the intensity of the fluorescence emitted by the sample
detector
detectors in fluorescence spectrophotometry
- photomultiplier tube (PMT)
- photodiode array (PDA) detector
records and displays the fluorescence spectra obtained from the sample
data output system
Analytical Steps in Fluorescence Spectrophotometry
- excitation
- emission
- detection and measurement
- spectral analysis
- quantification
- sample is exposed to light at a specific wavelength
- this energy is absorbed by the sample’s molecules, causing electrons to move to higher energy levels
excitation
excited electrons return to their ground state, they emit fluorescence at longer wavelengths
emission
- detector captures the emitted fluorescence, and its intensity is measured
- emitted light passes through a monochromator
- monochromator filters out unwanted background light
- filtered fluorescence reaches the detector
- detector converts the optical signal into an electrical signal
detection and measurement
by scanning the excitation wavelength across a range and recording the corresponding emission intensity, fluorescence spectrum is created
spectra analysis
intensity of the fluorescence signal can be related to the concentration of the fluorescent compound
quantification
fluorescence spectrophotometry application example
evaluate gamete and embryo functionality in animals and humans
studies how electricity flows and functions within living cells and organs
electrophysiology
