Chapter 17 - Fundamentals of Spectrophotometry Flashcards
What is colorimetry?
A procedure based on the absorption of light
What is spectrophotometry?
Any technique that uses light to measure chemical concentrations.
What is transmittance?
The fraction of original light that makes it through the sample.
What happens in a single-beam spectrophotometric experiment?
- light is passed through a monochromator from a light source
- the monochromator selects one wavelength
- the monochromatic light strikes a sample of length “b”
- the remaining light passed through the sample is measured by a detector
What are two examples of luminescence?
Fluorescence and phosphorescence
What is fluorescence?
The emission of a photon during a transition between states with the same spin quantum numbers (i.e. S1-> S0)
(Excited singlet state photon emission)
S0 is the ground electronic state
S1 is the lowest excited singlet electronic state
What is phosphorescence?
The emission of a photon during a transition between states with different quantum numbers (i.e. T1 -> S0)
(Excited triplet state photon emission)
S0 is the ground electronic state
T1 is the lowest excited triplet electronic state
What is stokes shift?
The difference in wavelength between the band maxima of the absorption and emission spectra of the same electronic transition
Why is luminescence more sensitive than absorption?
Luminescence is more sensitive than absorption because of the relative amount of light change.
What is an emission spectrum?
An emission spectrum is when the excitation wavelength is fixed and all the wavelengths of the emitted radiation are measured.
What is an excitation spectrum?
An excitation spectrum varies in excitation wavelengths and it measures one particular emission.
What are the advantages and disadvantages of fluorescence?
Advantages
- high sensitivity
- high selectivity
Disadvantages
- not all molecules fluoresce
- self quenches
What is self-quenching (fluorescence)?
Analyte molecules absorb too much light prior to reaching the sample cell region and the emission light is then absorbed by other analyze molecules before it can exit the sample cell
What are some applications of fluorimetry?
- air pollutants
- hydrocarbon energy sources (oil)
- food
- pharmaceuticals (drugs)
- agricultural (pesticides)
- biochemical and biomedical (GFP)
- LUMIER
- FRET
What is green fluorescent protein (GFP)?
It is a specialized protein in which emits fluorescence when exposed to excitation wavelength. The GFP gene can be expressed in other living organisms and it can be fused to non-fluorescent proteins as well.
What is FRET?
FRET also known as fluorescence resonance energy transfer is the transfer of energy from an excited donor chromophore to an acceptor chromophore, without associated radiation release.
Why is luminescence more sensitive than absorption?
Luminescence is more sensitive than absorption because of the relative amount of light change.
What is an emission spectrum?
An emission spectrum is when the excitation wavelength is fixed and all the wavelengths of the emitted radiation are measured.
What is an excitation spectrum?
An excitation spectrum varies in excitation wavelengths and it measures one particular emission.
What are the advantages and disadvantages of fluorescence?
Advantages
- high sensitivity
- high selectivity
Disadvantages
- not all molecules fluoresce
- self quenches
What is self-quenching (fluorescence)?
Analyte molecules absorb too much light prior to reaching the sample cell region and the emission light is then absorbed by other analyze molecules before it can exit the sample cell
What are some applications of fluorimetry?
- air pollutants
- hydrocarbon energy sources (oil)
- food
- pharmaceuticals (drugs)
- agricultural (pesticides)
- biochemical and biomedical (GFP)
- LUMIER
- FRET
What is green fluorescent protein (GFP)?
It is a specialized protein in which emits fluorescence when exposed to excitation wavelength. The GFP gene can be expressed in other living organisms and it can be fused to non-fluorescent proteins as well.
What is FRET?
FRET also known as fluorescence resonance energy transfer is the transfer of energy from an excited donor chromophore to an acceptor chromophore, without associated radiation release.
