Fluorescence Assay Flashcards
What is the basic principle of Fluorescence?
it its luminescence emiision of light from a substance
Where does emission takes place
at the excited state of the measured substance
Which is molecule can be used in fluorescence?
fluorescence (aromatic molecules)
what does the singlet state?
it is the lowest electronic energy level (stable configuration)
Does the singlet state showes flurophore?
NO, because of no excitation
What happend when light hit the molecule?
the Fluorophore absorbs the energy from the light, resulting in the excitation of the molecule to a higher energy level
Draw the jablonski-diagram
the exciation of the molecule
On what does the excitation level depends?
on the wavelength and the energy from the light source
what happend with the electrone at the higher energy level
it is in a unstable state, which leads to relaxation to a lowest energy level (s1)
What happend during the relaxation process?
it results in loss of energy
what is the excited lifetime?
it is the time at which the intercal concersion occur
what happend from s1-so state?
energy is being release and emitted as light
What is the emitted light
it is the fluorescence emission
compared the energy of both absorbt and emitted light
the emitted light is of lower energy than the absorb light
how is the energy level related to the wavlength?
lower energy, longer wavelenth
higher energy, shoter wavalength
what is the reason for the low energy of the emitted light?
it is because of the loss of engery during the relaxation process
What affects the intensity of the emitted light
it is affected by the illimination deviation to lower and higher wavelength
What is the stokes shift?
it is the difference between the excitation and emission maximal. in other words, the emission maximaum (longer wavelength) is is different from that of the absorb light (high energy, short wavelength)
On what dose the stokes shift depends?
it depends on the energy loss during the relaxation of the flurophore molecule.
which method can be used to determine the Limit of detection
Blank-value method and the Signal-to-noise ratio
defind the Blank-value method
the chosen concentration is 10 times lower than the lowest concentration in the calibration curve
Equation of the Blank-value method
LOD=3.3x Sd/m
What is the Limit of detection
it is the smallest amount of analyste in the sample that is assumed to be higher than in a blank sample.
When the S/N applied
it is applied tp procedures exhibiting baseline Noise.
How is the LOD with defind N/S defind
it is by comparing the minimum concentration at which the analyte can be detected from samples with knwon low concentration and blank samples
Equation of the S/N
S/N= 2H/h
why is the sensitivity of a Uv.is spectrometer easy to define?
it is because of the absoulte unit of measurement called the Absorbance value
LOD and LOQ
LOD: the smallest amount of the analyte in the sample which can be detected
LOQ: quantitation of the smallest amount of the analyte
Equation of LOQ
LOQ= 3 * LOD
Disadvantges of the Fluori
Non-linearity temparaure ph effects inner-filter effects quenching
Non-linearity Fluori
occured due to higher concentration of the fluorophore, saturated
Quenching in Fluori
when there is an interaction of the excited fluorohore molecule with uts surrounding which leads to a decrease of the intensity
Disadvantges of UV-Vis
Selectivity, low sensitivity
Selectivity UV-vis
no distintion between the samples of interest and present contamination which abort at the same wavelength
low sensitivity UV-vis
sensitivity is higher at a higher concentration, leading to additional steps to oncrease the concentration of the sample
