Direct and Indirect Analyses by Fluorescence Spectrophotometry: Quinine in Tonic Water Knowledge Check Questions

What happens during the excitation portion of fluorescence?

Electrons absorb energy/radiation and go up an energy level

Why is the fluorescence spectrum always at a longer wavelength than the absorption spectrum?

Fluorescence always occurs at the lowest state of the molecule after absorbance. However, absorbance excites molecules to a number of high energy states the one with the longest wavelength then happens to be that of the shortest for fluorescence.

Why does fluorescence spectroscopy provide a very low detection limit?

Fluorescence depends only on the power of the light source so high energy materials like lasers can be used.

Fluorescence is temperature dependent. Propose a reason for this. Hint: consider the phenomena that competes with fluorescence.

High temperatures could increase the transfer of energy from quinine to other substances in solution.

What is the primary limitation of fluorescence spectroscopy? What are the primary advantages of fluorescence?

Limitation- not all compounds fluoresce. Advantage- high sensitivity and specificity; fast and rapid diagnosis

What is the minimum number of filters/monochormators a fluorescence spectrophotometer contains? What are their purposes?

Two- one to excite the molecule and the other to measure the fluorescence

How does an effective quencher decrease the fluorescent intensity?

Quenchers have energy transferred to them from the quinine molecules.

What are some properties of an effective quencher, and what are the effective quenchers in this experiment?

Quenchers should be large or paramagnetic species chloride, bromide or iodide would make effective quenchers here and chloride is what will be used.

How can fluorescence quenching be used to quantify an analyte?

Quenching can be used to create a calibration curve of fluorescence intensity ratio vs quencher concentration

What is the difference between fluorescence and phosphorescence?

Fluorescence- emitting light by something that has absorbed light or radiation Phosphorescence- doesn't immediately re-emit the radiation

The fluorometer detector window is positioned at a right angle to the source window, rather than directly across. What is the advantage of this?

Minimizes interference of the light source with the fluorescence

What is quenching?

Process that decreases the fluorescence intensity of a substance

Name two ways a molecule can "give up" its extra energy when in an excited state.

Release excess energy as heat or light

How do you expect the fluorescence of quinine to change with the increasing concentration of chloride and why?

The fluorescence should decrease because quenchers dissipate the high energy state of quinine after absorption.

What happens after the excitation portion of fluorescence?

The particle gives off light (fluoresces)

What is a source used for fluorescent instruments?

Xenon arcs or lasers