L10 - spectroscopy Flashcards
define spectroscopy
use of electromagnetic radiation to characterise matter
shorter wavelength =
higher frequency
higher energy
longer wavelength =
lower frequency
lower energy
what is resonance spectroscopy
spectroscopy to detect the movements within atoms and molecules
the electromagnetic frequency used to detect this has to match the motion its detecting
eg electron spin
define constructive interference
two of the same frequencies in phase can combine to produce bigger frequency
define destructive interference
two of the same frequencies out of phase will cancel eachother out (180° out of phase)
define chromophore
‘system’ that can absorb electromagnetic radiation (typically in visible / uv range)
what do spectrometers do?
show the wavelengths absorbed/transmitted by a sample
what is UV-Vis absorbance spec
using UV light to detect how much of a molecule is present in a sample (molecule has to absorb UV/ visible light)
explain process of UV - Vis absorbance spec
- liquid sample containing substance of interest put into cuvette
- cuvette put into spectrophotometer and light shone through at certain frequencies
- beer lambert law used to calculate how much light absorbed
- absorbance used to calculate conc of material in sample
biological uses of UV - vis absorbance spec
measure concs of proteins in sample
some contain ring structures where the double bonds absorb certain wavelengths that can be detected by uv’-vis
what is infrared spec
the frequencies used interact with frequencies of bond vibrations to show what bonds are present in an unknown sample in order to identify it
how does infrared spec wavelengths compare to uv/visible
longer wavelengths (lower freq)
uses of infrared spec
identifying what substance is present in a sample by the bonds present
limitations of infrared spec
- bond interactions eg H bonds in water can interfere
2. peaks may overlap especially with macromolecules
what phase is used in UV-Vis spec
liquid
explain process of fluoresence spec
- light shone at cuvette containing fluorophore and detector placed 90° from it
- fluorophore absorbs light and emits back out at a different frequency
what is a fluorophore
substance that can absorb light and fluoresce
explain the excitation emission phenomenon
a fluorophore can absorb light at one frequency then emit it at a different frequency
is the emission or excitation wavelength longer?
emission
is the emission intensity as high as the excitation?
no
the emission is not as intense as the excitation
(not 100% of absorbed is emitted)
wave height of emission is smaller
define quantum yield
efficiency of light emitted
difference between emission height and excitation height
what is stokes shift?
difference between excitation wavelength and emission wavelength
applications of fluorescence spec
- detect single molecules
