Light scattering (Rayleigh and Mie).
- Lights scattering happens when EM waves encounter particles in air, Light waves cause a dipole moment, so electrons in atom vibrate, Cause them to emit light
- Rayleigh: Depends on wavelength (blue scattered more), When spacing is larger than wavelength there is no interference. Intensity of incident light = intensity of scattered light
- Mie: All wavelengths scatter equally, producing white light, when size of particle is on order of wavelength.
The Beer-Lambert law.
In dilute solution, if solvent doesn’t absorb in applied wavelength, absorption coefficient is proportional to concentration of solute. (Formula)
Properties of the absorption spectrum.
- Absorbance or transmittance (photon goes through something) as a function of wavelength
- Can be used to identify an element
- Absorption requires excitation of electron
Turbidimetry and nephelometry.
- Turbidemitry: Involved with measuring the amount of transmitted light, and calculating absorbed light by particles in suspension to determine conc. Of a substance.
- Nephelometry: At low intensity, measures scattered light. Proportional to conc. Amount of scattered light is much greater than transmitted, this method offers a higher sensitivity than turbidimetry.
- Both Dependent on: Number of particles, Size of particles.
Dynamic light scattering
- Method of analyzing solutions where hydrostatic diameter of particle can be measured.
- Light directed through sample —> Scattering occurs —> light intensity detected on other side —> intensity changes when particles in solution diffuse (brownian motion) —> speed of change depends on size of molecules —> information about intensity used to calculate diffusion coefficient.
Formula: D= k*t/6pinr
Measurement of the absorption spectrum.
- Absorbance vs wavelength of incident light.
- Absorption maxima
- Incident light must be at certain frequency
Energy levels of atoms and molecules: the Jablonski diagram
(Diagram)
Kasha’s rule when electron goes to closest energy level (internal conversion)
And intersystem crossing when going to Triplet or “Forbidden state”
Thermal radiation
- Transfer of heat using EM radiation
- Possible even in Vacuum
- Everything over 0K radiates thermal radiation
Planck’s radiation law
- Studied emission spectrum of black bodies
- Energy emitted resulted from vibration of atoms within the material
- Vibrational energies have discrete values, 1, 2, 3 , never in between.
- E2 - E1 = e = hf
- 6.626 x 10^-34
Light sources based on thermal radiation
- Sun, lightbulbs, heated metal.
Absolute black body
- Ideal, theoretical body which absorbs all radiation incident on it and reemits it.
- Model can be created from closed metal cavity with hole drilled so radiation entering can not easily escape, so absorbed completely.
- Stefan-Boltzmann law describes that emittance of a black body is proportional to the fourth power of the temperature. M black = o T4
Emission spectrum of the absolute black body.
- Emission is in all wavelength spectrum
- Wien’s law: Maximum radiation is in wavelength that is inversely proportional to temperature.
- At low temp, black body is dark, most energy radiated is infra-red
- When temp increases it glows red first, then yellow, then white/blue.
Medical applications of thermal radiation
- Thermography: Test using infrared camera to detect heat patterns and blood flow in body tissues. DITI is the type of thermography used to diagnose breast cancer. (Thermotropic crystals used)
Kirchhoff’s law
- A body which radiates more thermal energy also absorbs thermal energy to a higher extent.
- Ratio between radiant emittance and absorption coefficient is constant with a narrow wavelength range.
The Stefan-Boltzmann law.
Describes that the emittance of a black body is proportional to the 4th power of the temperature.
M black = o T^4
Wien’s displacement law
- The black body radiation curve for different temp peaks at a wavelength inversely proportional to the temperature.
Luminescence: excitation and relaxation.
- Emission of excess energy from excited electrons in form of light.
- Types of excitation: Thermo, bio, photo electro.
- Process: Absorption of external energy causes excitation and emission of energy in the form of light.
- Types of relaxation: Fluorescence, Phosphorescence
Kasha’s rule
- The excited molecule first reaches the lowest vibrational level S1
- Internal conversion
- Photon emission occurs going to ground state.
Fluorescence
- If the luminescence stops as excitation stops
Phosphorescence
- During transition from triplet state to ground state
- Slower luminescence than fluorescence.
Luminescence spectra
- Atoms: Line spectra (Medium pressure Hg lamp)
- Molecules: Band spectrum, (High pressure Hg lamp)
Stokes-shift
Shift difference between peak absorption and peak emission due to loss of energy in the form of heat.
The fluorescence spectrometer
Device which shines light through a sample, measuring excitation spectrum and resulting emission spectrum.
- Parts: Xe lamps, Excitation monochromator, sample cell, emission monochromator, photon detector.
(FRET) Fluorescence Resonance Energy transfer
Energy transfers from donor molecules without emission, to acceptor molecules when they form dipole-dipole interactions.
1) They have to be in the right orientation
2) They have to be really close
3) Spectral overlap between donor and acceptor
Applied to protein-protein interactions
