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Quality Control > Fluorescence Assay > Flashcards

Fluorescence Assay Flashcards

1
Q

What is the basic principle of Fluorescence?

A

it its luminescence emiision of light from a substance

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2
Q

Where does emission takes place

A

at the excited state of the measured substance

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3
Q

Which is molecule can be used in fluorescence?

A

fluorescence (aromatic molecules)

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4
Q

what does the singlet state?

A

it is the lowest electronic energy level (stable configuration)

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5
Q

Does the singlet state showes flurophore?

A

NO, because of no excitation

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6
Q

What happend when light hit the molecule?

A

the Fluorophore absorbs the energy from the light, resulting in the excitation of the molecule to a higher energy level

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7
Q

Draw the jablonski-diagram

A

the exciation of the molecule

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8
Q

On what does the excitation level depends?

A

on the wavelength and the energy from the light source

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9
Q

what happend with the electrone at the higher energy level

A

it is in a unstable state, which leads to relaxation to a lowest energy level (s1)

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10
Q

What happend during the relaxation process?

A

it results in loss of energy

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11
Q

what is the excited lifetime?

A

it is the time at which the intercal concersion occur

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12
Q

what happend from s1-so state?

A

energy is being release and emitted as light

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13
Q

What is the emitted light

A

it is the fluorescence emission

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14
Q

compared the energy of both absorbt and emitted light

A

the emitted light is of lower energy than the absorb light

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15
Q

how is the energy level related to the wavlength?

A

lower energy, longer wavelenth

higher energy, shoter wavalength

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16
Q

what is the reason for the low energy of the emitted light?

A

it is because of the loss of engery during the relaxation process

17
Q

What affects the intensity of the emitted light

A

it is affected by the illimination deviation to lower and higher wavelength

18
Q

What is the stokes shift?

A

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)

19
Q

On what dose the stokes shift depends?

A

it depends on the energy loss during the relaxation of the flurophore molecule.

20
Q

which method can be used to determine the Limit of detection

A

Blank-value method and the Signal-to-noise ratio

21
Q

defind the Blank-value method

A

the chosen concentration is 10 times lower than the lowest concentration in the calibration curve

22
Q

Equation of the Blank-value method

A

LOD=3.3x Sd/m

23
Q

What is the Limit of detection

A

it is the smallest amount of analyste in the sample that is assumed to be higher than in a blank sample.

24
Q

When the S/N applied

A

it is applied tp procedures exhibiting baseline Noise.

25
Q

How is the LOD with defind N/S defind

A

it is by comparing the minimum concentration at which the analyte can be detected from samples with knwon low concentration and blank samples

26
Q

Equation of the S/N

A

S/N= 2H/h

27
Q

why is the sensitivity of a Uv.is spectrometer easy to define?

A

it is because of the absoulte unit of measurement called the Absorbance value

28
Q

LOD and LOQ

A

LOD: the smallest amount of the analyte in the sample which can be detected

LOQ: quantitation of the smallest amount of the analyte

29
Q

Equation of LOQ

A

LOQ= 3 * LOD

30
Q

Disadvantges of the Fluori

A 
Non-linearity
temparaure
ph effects
inner-filter effects
quenching
31
Q

Non-linearity Fluori

A

occured due to higher concentration of the fluorophore, saturated

32
Q

Quenching in Fluori

A

when there is an interaction of the excited fluorohore molecule with uts surrounding which leads to a decrease of the intensity

33
Q

Disadvantges of UV-Vis

A

Selectivity, low sensitivity

34
Q

Selectivity UV-vis

A

no distintion between the samples of interest and present contamination which abort at the same wavelength

35
Q

low sensitivity UV-vis

A

sensitivity is higher at a higher concentration, leading to additional steps to oncrease the concentration of the sample

Quality Control (7 decks)

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