Lecture 9 Light Spectroscopy Flashcards
Electromagnetic radiation spectrum
Visible light is a small fraction of the electromagnetic spectrum
Photoenergy E=hc/gamma= hv
Multiply by Avogadro’s consonant for joules per mol
h= Planck’s constant (6.26x10-³⁴ Js)
c= speed of light in a vacuum (2.998x10⁸ms-¹)
Gamma= wavelength in m, Vis freq in Hertz (s-1)
A photon is both a wave and a particle
- shorter the wavelength, higher the quantity of energy carried, it behaves like a particle under norm conditions
- long wavelengths non-ionising and not damaging (short waves opposite)
Radiation from the sun
Energy of light from sun as a function of a wavelength as a distribution curve
X wavelength/ y spectral dist (W/m²)
Curve starts off as a nice curve (“black body” radiation for an object at 5500K)
but the atmosphere absorbs certain wavelengths strongly so that only some reach the surface
Even less wavelengths penetrate the oceans so they fade rapidly with depth.
Note: Ozone O3 absorbs UV strongly protecting us from more severe sun burn and skin cancer
Absorption of photons in visible and UV light
Results in excitation of electrons to higher energy levels.
Absorption of a photo of the correct energy (wavelength) leads to excitation of the electron to a higher energy level (green arrows)
If the photon has too low an energy (too long a wavelength) absorption does not occur
If the photon has too high an energy (too short a wavelength) absorption does not occur.
Absorption requires that the photons energy exactly matches the energy gap between two energy levels.
Can be shown on a Jablonski diagram: energy levels are fixed by quantum mechanics, photons of specific energy (wavelength) are required to allow excitation.
Molecular electronic transitions
Take place when electrons in a molecule are excited from one energy level to a higher energy level.
The energy change associated with each transition is determined by chemical composition and structure of the molecule.
This is represented by vertical dist. In Jablonski diagram.
Molecular electronic transitions determine many molecular properties such as colour.. this is because the distance apart of the energy levels determines the energy of the photon that is absorbed and thus it’s wavelength.
Each compound has a characteristic spectrum of absorption due to its molecular electronic transitions. Changes in ionisation of structure affect these transitions.
Fine details due to the vibrational levels are smoothed out in solution
Excitation of electrons in molecules like chlorophyll are the starting point for photosynthesis
How absorption spectra are measured for compounds in solution: spectrometers
Single beam/ diode array spectrometer
Single: light passes through entrance slit, through monochromator, exit slit, sample then detector
Diode array: light passes through sample, entrance slit, monochromator then detector
Diffraction grating of monochromator
Determines the wavelength of light (gamma) passing to the sample. A simple prism is similar
Absorption spectrum of a white light
White light spectrum from LED torches are made up of blue red and green light emitters
How are absorption spectra measured?
The colour of a compound in a solution is determined by absorption of light whose wavelength is the complementary colour
A colour spectrometer shows the absorption spectrum of a sample e.g. a blue solution absorbs red light
Absorbance and concentration: Beer -Lambert law/equation (see end for equation)
Law gives a linear relation between absorbance and concentration
Absorbance A= epsilon c L
A= absorbance
Epsilon= molar absorption coefficient with units M-¹ or cm-¹
L= path length of sample - path length of cuvette in which sample is contained - conventionally expressed in cm
c= conc. of compound in solution expressed in Moles
Epsilon can be estimated from a standard curve or looked up in ref. Literature for a given compound
Epsilon is normally specified as a wavelength usually an absorption maximum for a compound under defined conditions in solution
The higher epsilon is the more strongly compound absorbs light
Beer Lambert law breaks down at high conc due to interactions between absorbing molecules - linear relationship flattens
The amount of radiation absorbed may be measured in a no. of ways :
Transmittance: T=l/ Io
= Light intensity out/light intensity in
Or
% transmittance %T=100T=100xI/Io
Or
Absorbance A=log10 Io/I
Fluorescence
Normally after an electron is excited by a photon to a higher level energy is transferred into vibrational and other molecular motion (e.g. heat) causing it to return to its original energy level
Sometimes an electron gets ‘stuck’ at a higher energy level and has to lose the energy by emitting a photon
Because some of the initial energy is already dissipated by molecular motion the emitted photon always has lower energy than the absorbed photon
Lower energy so longer wavelength
Whether or not fluorescence occurs depends on the structure of the compound.
How is fluorescence measured?
By a fluorescence spectrometer
Light source >
monochromator>
Determines wavelength of excitation gamma ex
sample>
> fluorescence monochromator ( light emission measured at right angle to the incident beam to prevent detection of transmitted light). The fluoro monochromator determines the wavelength of emission gamma f. It prevents scattered light at excitation wavelength from being detected.
> Recorder
There is no “molar fluorescence constant” or “fluorescence Beer Lambert Law” because detection is instrument - dependent.
However for a given set-up emission is proportional to conc. Of emitting compound.
Fluorescence microscope
Has become the most important tool in the cell biologists repertoire - using probes containing fluorescent labels allows specific cell structures to be studied in vivo
