Fluorescence Spectrophotometry

Question 1

method used to investigate molecular and atomic interactions by examining the fluorescence emitted from a sample

Answer 1

fluorimetry and spectrofluorimetry

Question 2

type of luminescence caused by photons exciting a molecule, raising it to an electrnic excited state

Answer 2

fluorescence

Question 3

method used to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution

Answer 3

Spectrophotometry

Question 4

• process begins with the absorption of photon by a molecule, called the fluorophore, which results in the promotion of an electron to a higher energy state
• aborbed energy excites the molecule from its ground state to an excited state

Answer 4

excitation

Question 5

absorbs photons and emits photons of lower energy in return

Answer 5

fluorophore

Question 6

as excited molecule returns to the ground state, it emits a photon of lower energy than the absorbed photon

Answer 6

emission

Question 7

• illustrates the transition of electronic states during fluorescence
• shows the energy levels and the absorbance spectrum of a typical fluorescent molecule

Answer 7

Jablonski Diagram

Question 8

• refers to the average time or duration a fluorophore spends in the excited state before releasing a photon and returning to its ground state
• can vary from picoseconds to hundreds of nanoseconds depending on the fluorophore

Answer 8

fluorescence lifetime (FLT)

Question 9

General Componenets of fluorescence spectrophotometry

Answer 9

1. light source
2. monochromator
3. sample holder
4. detector
5. data output system

Question 10

can emit radiation in the ultraviolet, visible, and near-infrared wavelengths

Answer 10

light source

Question 11

selects a specific wavelength of light from the broad spectrum emitted by the light source

Answer 11

monochromator

Question 12

Parts of the monochromator

Answer 12

1. entrace slit
2. collimating lens
3. diffraction grating or prism
4. exit slit

Question 13

controls the width of the incident light beam

Answer 13

entrance slit

Question 14

converts divergent light rays into parallel rays

Answer 14

collimating lens

Question 15

selects a narrow range of wavelengths for excitation

Answer 15

diffraction grating or prism

Question 16

determines the bandwidth of the selected excitation wavelength

Answer 16

exit slit

Question 17

• where the sample to be analyzed is placed
• usually quartz cuvette or a microplate, designed to hold the sample securely and allow the excitation light to pass through it

Answer 17

sample holder

Question 18

sample holders are usually what?

Answer 18

• quartz cuvette or
• microplate

Question 19

measures the intensity of the fluorescence emitted by the sample

Answer 19

detector

Question 20

detectors in fluorescence spectrophotometry

Answer 20

• photomultiplier tube (PMT)
• photodiode array (PDA) detector

Question 21

records and displays the fluorescence spectra obtained from the sample

Answer 21

data output system

Question 22

Analytical Steps in Fluorescence Spectrophotometry

Answer 22

1. excitation
2. emission
3. detection and measurement
4. spectral analysis
5. quantification

Question 23

• sample is exposed to light at a specific wavelength
• this energy is absorbed by the sample’s molecules, causing electrons to move to higher energy levels

Answer 23

excitation

Question 24

excited electrons return to their ground state, they emit fluorescence at longer wavelengths

Answer 24

emission

Question 25

• detector captures the emitted fluorescence, and its intensity is measured
• emitted light passes through a monochromator
• monochromator filters out unwanted background light
• filtered fluorescence reaches the detector
• detector converts the optical signal into an electrical signal

Answer 25

detection and measurement

Question 26

by scanning the excitation wavelength across a range and recording the corresponding emission intensity, fluorescence spectrum is created

Answer 26

spectra analysis

Question 27

intensity of the fluorescence signal can be related to the concentration of the fluorescent compound

Answer 27

quantification

Question 28

fluorescence spectrophotometry application example

Answer 28

evaluate gamete and embryo functionality in animals and humans

Question 29

studies how electricity flows and functions within living cells and organs

Answer 29

electrophysiology