Spectrometry Flashcards
(24 cards)
Definition: Spectroscopy
The study of the interaction of matter with light (i.e electromagnectic radiation)
Definition: Spectrometry
The method used to acquire a spectrum
Definition: Spectrophotometry
Spectrometry in the UV-visible-near infrared range
Used most in biochemistry labs
Applications of Spectrophotometry
- analysis of materials
- quantitation of components, including in complex mixtures
- time-resolved (monitoring of rates of changes of reactants and products)
What is the Planck-Einstein relation
E= hv
Energy = Plank constant x frequency
J=J.s s^-1
What is the Planck constant
~6.626x10^-34 J.s
Which has the higher energy X-rays or Radio waves
Xrays
Which has the higher frequency X-rays or Radio waves
Xrays
Which has a longer wavelength X-rays or radio waves
Radio waves
Energy and frequency are___________
Energy and wavelength are _______ proportional
- proportional
2. inversely
What equation relates wavelength, speed of light and frequency
λ= c/v m = ms-1 / s-1
What is the range of the visible spectrum
~700nm - ~400 nm
What is an electronic transiton
When photons in the UV/visible/near-IR range cause electrons to move from one orbital to another of higher energy
When does electronic transition occur
When the E of the absorbed photons is equal to the energy required to move the electrons between the orbitals
What happens when you place a substance in a light path
It results in a band of absorption at specific wavelengths by a substance
_________ ,___________, __________ of the bands of absorption are characteristic of the absorbing substance
Wavelength
Shapes
Intensities
Haem groups have strong bands in the ____ and ____
Wavelengths depends on the _____and on the _____ state e.g _____, _____ and _____
400-450 nm and 550-610 nm
haem type
chemical
oxidizes, reduced, ligand-bound
What colour is oxidised Haemoglobin (Fe3+)
Brown
What colour is reduced Haemoglobin (Fe2+)
Purple
What colour is oxygen-bound, reduced Haemoglobin (Fe2+ … O2)
Red
Wavelengths:
Tyrosine
Tryptophan
Nucleic acid bases
280nm
280nm
260nm
Proteins with known number of Try and Trp can be quantitated from their absorbance at 280nm
What is used for quantitative analysis of absorption
Spectrophotometer
What are the basic components of a typical spectrophotometer
Light source: Halogen lamp/ tungsten filament λ>300nm
Deuterium arc λ ~ 350-200nm (for Tyr, Trp, DNA)
Dispersion device: prism/ diffraction grating to separate the different wavelengths
Exit slit: Select specific wavelengths with a narrow slit
Sample: Within a cuvette (below visible light quartz cuvette)
Detector: T measure the intensity of light. Converts a stream of photos into a stream of electrons.
What can absorbance spectroscopy be used for
Absorbance bands- Qualitative.
Absorbance spectrum- Measure the absorbance at each wavelength, plot absorbance vs wavelength- Quantitive
