G6- Fluorescence Spectrophotometry Flashcards
• also known as “fluorimetry” or “spectrofluorometry”
• a method used to investigate the molecular and atomic interactions by examining the fluorescents emitted from a sample.
Fluorescence spectrophotometry
Fluorescence spectrophotometry also known as fluorimetry or spectrofluorometry is a method used to investigate the ___ and ___ interactions by examining the fluorescents emitted from a sample
molecular and atomic interactions
is a type of luminescence caused by photons exciting a molecule raising it to an electronic excited state
fluorescence
is a 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
The process begins with the absorption of ___ by ____ which result in the promotion of an electron to a higher energy state.
photon ( light)
molecule ( fluorophore
absorb energy excites the molecule from its ground state to an excited state. As the excited molecule returns to the ground state it emits a photon of ____ (corresponding to a longer wavelength) than the absorb photon
lower energy
illustrates the transition of electronic states during fluorescence. It shows the energy levels in the absorbent spectrum of a typical fluorescent molecule
Joblonski diagram
Joblonski diagram illustrates the transition of ____ states during fluorescence.
electronic
•Refers to the average time/duration of fluorophore spends in the excited state before releasing a photon and returning to its ground state.
•It can vary from picoseconds to hundereds of nanoseconds depending on the fluorophore.
Fluorescence lifetime
excited state formula
I(t) = Be^t/T (di jud siya T pero ana iyang na nawong)
t
T
B
??
t- time
T- fluorescence lifetime
B- pre-exponential factor
THE INSTRUMENT IS FITTED WITH A ___ __ THAT CAN EMIT RADIATION IN THE ULTRAVIOLET, VISIBLE, AND NEAR INFRARED WAVELENGTHS
LIGHT SOURCE
SELECT A SPECIFIC WAVELENGTH OF LIGHT FROM THE BROAD SPECTRUM EMITTED BY THE LIGHT SOURCE
MONOCHROMATOR
MONOCHROMATOR is composed of:
entrance 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
which determines the bandwidth of the selected excitation of wavelength
Exit slit
It is where the sample to be analyzed is placed. It is usually quartz cuvettes or a microplate designed to hold the sample securely and allow the excitation light to pass through it
Sample holder
Sample holder is usually ___, ____ or a __ designed to hold that the sample securely and allow the excitation light to pass through it
quartz
cuvettes
microplate
• measures the intensity of the fluorescence emitted by the sample. •Typically a photo multiplier tube or pmt and photodiode array or pda detector
Detector
records and displays the fluorescence spectra obtained from the sample
Data output system
GENERAL COMPONENTS
Light source
Monochromator
Sample holder
Detector
Data output system
Analytical steps
Excitation
Emission
DETECTION AND MEASUREMENT
SPECTRAL ANALYSIS
QUANTIFICATION
the 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
the excited electrons return to their ground state, they emit fluorescence at longer wavelengths
Emission
the ___ captures the emitted fluorescence, and its intensity is ___.
Detector
measured
What happens in detection and measurement
The emitted light passes through a monochromator
• The monochromator filters out unwanted background light.
• The filtered fluorescence reaches the detector.
• The detector converts the optical signal into an electrical signal.
by scanning the excitation wavelength across a range and recording the corresponding emission intensity, fluorescence spectrum is created
SPECTRAL ANALYSIS
the intensity of the fluorescence signal can be related to the concentration of the fluorescent compound
QUANTIFICATION
In Jablonski diagram, the ___ line is the ground state (S0), and ___ lines represent excited states (S1, S2, etc.)
Lowest lines
Higher lines
• After reaching an excited state, the molecule rapidly loses some energy through ___ ___, which involves non-radiative transitions to lower vibrational levels within the same electronic state
• shown by downward wavy lines in Jablonski diagram
vibrational relaxation
In some cases, the molecule can transition from an excited singlet state (S1) to a triplet state (T1) through ___ ___ (depicted by a crossing arrow).
intersystem crossing
refers to the range of wavelengths of light that are allowed to pass through the exit slit
Bandwidth
