Fluorescence Spectroscopy

Question 1

What is the definition of fluorescence?

Answer 1

The property of some atoms and molecules to absorb light of a particular wavelength and to subsequently emit light of a longer wavelength after a brief interval

Question 2

What is the definition of phosphorescence

Answer 2

Another form of photoluminescence but with much longer excited state lifetimes.

Question 3

What are the principles behind fluorescence spectroscopy?

Answer 3

There are 3 important events that are separated by several orders of magnitude of time.

Question 4

What is the first event?

Answer 4

Excitation of a susceptible molecule by an incoming photon (10^-15 seconds)

Question 5

What is the second event?

Answer 5

Vibrational relaxation of excited state electrons to the lowest vibrational level of the excited state (10^-12 seconds)

Question 6

What is the third event?

Answer 6

Emission of a longer wavelength photon and return of the molecule to the ground state (10^-9 seconds)

Question 7

What is the definition of vibrational relaxation?

Answer 7

Excited electrons transition from a higher excited energy level to a lower vibrational level within the same electronic state

Question 8

What is the definition of internal conversion?

Answer 8

Excited electrons transition from a vibrational level in one state to another vibrational level in a lower electronic state

Question 9

What is intersystem crossing?

Answer 9

a transition between electronic states of a different spin multiplicity, or in other words from the excited singlet state to an excited triplet state

Question 10

What is phosphorescence in this context?

Answer 10

A radioactive transition from an excited triplet state to a singlet ground state

Question 11

What qualities do we want in an ideal fluorescent molecule?

Answer 11

We want a high extinction coefficient, a high fluorescence quantum yield and a large Stokes Shift

Question 12

What is the definition of extinction coefficient?

Answer 12

A measurement of light absorption capacity

Question 13

What is meant by ‘fluorescence quantum yield’?

Answer 13

The ratio between the number of fluorescence protons emitted and the number of photons absorbed

Question 14

What is meant by a ‘Stokes Shift’? and why do we want a high Stokes Shift?

Answer 14

Stokes Shift is the difference between the peak excitation and the peak emission wavelengths. We want a large Stokes Shift so we can reduce the interference between absorption and emission

Question 15

What factors can influence fluorescence intensity?

Answer 15

Quenching, concentration, temperature, pH and inner filter effects

Question 16

Why does quenching affect fluorescence intensity?

Answer 16

Interactions between the excited state fluorophore and its surroundings are decreased

Question 17

Why do we quench?

Answer 17

to reduce the impact of the excipients of a tablet on the reading

Question 18

What processes can be involved in quenching?

Answer 18

Collisional quenching, excited state quenching, energy transfer, and complex formation

Question 19

How does concentration affect fluorescence intensity?

Answer 19

At high concentrations the intensity of excitation light is not uniform throughout the sample. In addition, some emitted light can be reabsorbed by other unexcited molecules, which can cause non-linearity between concentration and fluorescence

Question 20

How does temperature affect fluorescence intensity?

Answer 20

Quantum efficiency decreases with increasing temperature. The increased frequency of collisions between the fluorophores and solvent molecules at higher temperatures increases the chance of deactivation

Question 21

How does pH affect fluorescence intensity?

Answer 21

Wavelength and emission intensity are different for protonated and unprotonated forms

Question 22

How does ‘inner filter effects’ impact fluorescence intensity?

Answer 22

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