Fluorescence

Question 1

What is fluorescence?

Answer 1

Fluorescence is the emission of light by a molecule after it absorbs photons, causing excitation and subsequent relaxation to its ground state.

Question 2

How is fluorescence different from phosphorescence?

Answer 2

Fluorescence is almost instantaneous and stops when the light source is removed, while phosphorescence has a delayed emission, leading to an “afterglow.”

Question 3

What are the three main steps in fluorescence?

Answer 3

Excitation: Photon absorption excites electrons (10⁻¹⁵ s).
Vibrational relaxation: Energy loss as heat within the same electronic state (10⁻¹² s).
Emission: Photon release as the molecule returns to the ground state (10⁻⁹ s)

Question 4

What is a Jablonski diagram?

Answer 4

A diagram that illustrates the transitions of electrons during excitation, relaxation, and emission in fluorescence.

Question 5

What is the Stokes shift?

Answer 5

The difference between the excitation and emission wavelengths, indicating energy lost as heat.

Question 6

What is the Mirror Image Rule?

Answer 6

For many fluorophores, the fluorescence emission spectrum mirrors the absorption spectrum due to similar vibrational energy spacing in ground and excited states.

Question 7

What structural features make a molecule fluorescent?

Answer 7

Conjugated double bonds (-C=C-).
Cyclic/aromatic rings.
Increased rigidity in structure.

Question 8

How does rigidity affect fluorescence?

Answer 8

Rigid molecules lose less energy through non-radiative processes, leading to higher fluorescence efficiency.

Question 9

How is fluorescence spectroscopy used in pharmaceutical analysis?

Answer 9

Qualitative: Identifying fluorescent agents and tracking drug-target interactions.
Quantitative: Measuring fluorophore concentrations (proportional to fluorescence intensity).

Question 10

What are the advantages of fluorescence spectroscopy?

Answer 10

High sensitivity (100x more than UV).
Specificity: Can detect fluorescent drugs in the presence of non-fluorescent excipients.

Question 11

How does concentration affect fluorescence?

Answer 11

High concentrations can cause reabsorption or inner filter effects, leading to non-linearity between concentration and fluorescence intensity.

Question 12

What is quenching in fluorescence?

Answer 12

A decrease in fluorescence intensity due to interactions with quenching agents (e.g., collisional quenching, energy transfer, or complex formation).

Question 13

How does temperature affect fluorescence?

Answer 13

Higher temperatures increase molecular collisions, decreasing quantum efficiency and fluorescence intensity.

Question 14

What are the limitations of fluorescence spectroscopy?

Answer 14

Limited to fluorescent molecules.
Temperature-sensitive.
Self-quenching at high concentrations.

Question 15

Why are fluorescent intensities measured in arbitrary units?

Answer 15

They must be compared to a standard solution as fluorescence is relative, not absolute.

Question 16

Which of the following best describes fluorescence?
A. Absorption of light without emission.
B. Emission of light after excitation, occurring with a delayed “afterglow.”
C. Immediate emission of light after photon absorption.
D. Emission of light without requiring a light source.

Answer 16

C

Question 17

What is the Stokes shift in fluorescence spectroscopy?
A. The time delay between excitation and emission.
B. The distance between excitation and emission wavelengths.
C. The overlap between excitation and emission spectra.
D. The energy released as heat during vibrational relaxation.

Answer 17

B

Question 18

Which of the following structural features increases a molecule’s fluorescence?
A. Increased flexibility of bonds.
B. Presence of cyclic and conjugated structures.
C. Lack of double bonds.
D. Low molecular rigidity.

Answer 18

B

Question 19

What is the purpose of the Jablonski diagram in fluorescence?
A. To measure fluorescence intensity.
B. To illustrate molecular rigidity in fluorescence.
C. To describe transitions between energy states during fluorescence.
D. To explain the difference between fluorescence and phosphorescence.

Answer 19

C

Question 20

Which factor is most likely to reduce fluorescence intensity?
A. Low concentration of the fluorophore.
B. High quantum efficiency.
C. Collisional quenching.
D. Large Stokes shift.

Answer 20

C

Question 21

Which process occurs during vibrational relaxation?
A. Emission of a photon as the molecule returns to the ground state.
B. Transition between electronic states with different spin orientations.
C. Energy release as non-radiative heat within the same electronic state.
D. Excitation of an electron to a higher vibrational level.

Answer 21

C

Question 22

Which of the following is true about phosphorescence?
A. It emits light only while the light source is on.
B. It involves a spin-neutral relaxation process.
C. It has a longer lifetime due to triplet state involvement.
D. It occurs in molecules with no conjugated structures.

Answer 22

C

Question 23

What is an example of a limitation in fluorescence spectroscopy?
A. High sensitivity compared to UV spectroscopy.
B. Specificity for fluorescent molecules.
C. Temperature dependence of quantum efficiency.
D. Ability to detect low concentrations of analytes.

Answer 23

C

Question 24

Which method can be used to analyze non-fluorescent drugs?
A. Using a spectrophotometer without derivatization.
B. Reacting the drug with a fluorophore to create a fluorescent derivative.
C. Lowering the temperature to induce fluorescence.
D. Analyzing the drug in its non-fluorescent form using UV spectroscopy.

Answer 24

B

Question 25

What is the significance of a large Stokes shift?
A. It increases fluorescence self-quenching.
B. It makes it easier to distinguish emission from excitation light.
C. It decreases the fluorescence intensity of a fluorophore.
D. It increases reabsorption of emitted light.

Answer 25

B