Lecture 1: Scattering Flashcards
<p>What is scattering? </p>
<p>Scattering is when radiation hits a particle and is re-emitted at the same wavelength.
• We can see scattering in clouds, smoke, milk etc.
• Scatterers behave like driven oscillators.
• The intensity of scattering depends on size (RG and MW) and concentration of the scatterers.
• RG is the radius of gyration. It is related to particle dimensions.
• We can gain information about the shape of particles in solution and their motion.
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<p>How can we use MALS and SEC-MALS?</p>
<p>MALS stands for multiangle light scattering. It is often used in tandem with size exclusion chromatography.
• MALS uses the scattering of a beam of monochromatic light.
• MALS is often carried out after SEC.
• We can use MALS to find stoichiometry.
• The scattering intensity is proportional to the MW and concentration.
• λ is larger than RG¬.
• SEC is used to separate analytes.
• MALS can show molar mass and size of proteins.
• It can also be used to determine protein-protein binding.
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<p>How can we use SAXS and SANS?</p>
<p>SAXS and SANS are used to give detailed information about the shape of a molecule.
• SAXS and SANS both use a wavelength which is lower than RG¬. X-rays and neutrons (λ around 0.1 nm) fulfil this requirement.
• SAXS and SANS give a plot of lnI vs Q (Q is related to the scattering angle).
• Replotting, the very small angle data gives a Guinier plot (lnI vs Q2) that is linear. The slope is RG and the X-intercept is MW.
• Replotting all data gives a Kratky plot (I* Q2 vs Q). This plot reports on flexibility.
• At larger angles, the plot gives information about particle shape. This requires a Fourier Transform. We can calculate protein shapes at an effective resolution of about 30 Angstroms.
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<p>How can SANS be used for contrast scattering? </p>
<p>Neutron scattering can be different for different isotopes. this can be exploited experimentally.
• Scattering depends on the cross section of a nucleus.
• 1H has negative B while 2H has positive B.
• The scattered waves are 180 degrees out of phase.
• We can use contrast matching, where we adjust the solvent scattering cross section by changing the ratio of water and heavy water.
• We can match the solvent background to various macromolecular components in a solution.
• We can even selectively deuterate parts of a macromolecule and measure this with contrast matching.
• For example, SANS was used to identify protein positions in the 30S ribosomal particle.
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<p>What is dynamic light scattering? </p>
Dynamic light scattering is used to analyse the motion of macromolecules in solution.
• Static scattering is elastic. The frequency in and out are the same.
• If the scatterer moves than the frequency will be spread by the Doppler effect.
• Diffusion coefficients are measured by DLS.
• They can give information on sample size and aggregation.
• A laser is shone at a small volume. As molecules flow in and out of this volume, this causes changes in the intensity of scattered light.
• The rate of diffusion is related to the diffusion coefficients of the particles involved (DT). Large particles move more slowly.
• DT can be used to calculate the hydrodynamic radius (RH). RH depends on the shape and size of the scattering particle. It cannot give an accurate measurement of mass.
• We can use DLS to study a sample with multiple species or aggregates and get analysis of a sample with a broad distribution of species.
• Scattering intensity is proportional to mass. It can easily detect small amounts of high mass species. DLS can therefore be used to tell how homogenous a sample is by mass.
• DLS is easy and it requires little sample.
<p>What is surface plasmon resonance?</p>
Surface plasmon resonance is a technique which can be used to find the rate constants and dissociation constants of a ligand and its binding partner.
• It works based on the production of an evanescent wave.
• An evanescent wave forms at the boundary interface when an EM wave undergoes total internal reflection.
• Evanescent waves are electromagnetic waves that have an exponential decay in intensity as they move away from the interface.
• The wave causes oscillation (surface plasmon) of electrons in a thin metal film such as gold.
• At a specific incident angle, the electrons will undergo surface plasmon resonance. The reflected light will have a decreased intensity.
• Changes in material immobilised near the film will cause the SPR angle to change in proportion to the change in mass.
• The material can be immobilised directly (thiols, aldehydes or amides) or indirectly (antibodies, streptavidin and biotin).
• We must consider which materials we want to be prey and bait.
• We can plot RU (response units) on the X-axis and RU/[ligand] to give a slope of -1/KD.
• By plotting RU against time we can find the on rate constant and the off rate constant.
<p>What are the different types of scattering?</p>
<p>• If λ > RG then Rayleigh scattering is isotropic (same along all axes) and its intensity depends on size and amount.
• When λ ~ RG or λ < RG we get interference between scattered waves from different regions of a particle. The scattered intensity decreases with angle in a manner that depends on shape.
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How do total internal reflection and evanescent waves work?
TIR is a phenomenon exploited by SPR.
• When light moves from one medium to another, it is refracted.
• At a critical angle it will reflect and have a refraction angle of 90 degrees.
• We can calculate this with Snell’s law.
