Light Scattering Flashcards
How does light scattering happen
When the refractive index of a medium changes due to bubbles/particles in a liquid , droplets in a gas
When does Rayleigh scattering happen
What type of scattering is it
What is it proportional to
When the diameter of the particles scattering the light are «_space;the wavelength of the light
Eleastic scatteirng because is has the same wavelength for the input light and the output light (wavelength doesn’t change, same NRG going in and same energy going out)
Proportional to the molar mass of the particle and 1/Lambda^4
What does Rayleigh scattering cause
The blue colour in the sky during the day
In measuring light scattering what is actually being measured
What is static light scattering
the detector measures the light from the side not directly across from the light source
Can still measure across and that gives turbidity (OD) of the sample (how much loss of light there is in that direction due to scattering from sample turbidity)
Static: The average amount of light scattered
Rayleigh ratio equation on sheet
On sheet
What can the Rayleigh ratio be used for
What is special about the equation for find MW
can find the average molecular weight only when the molcule is much smaller than the wavelength
MW= R theta/ K c
The concentration for your sample needs to be very high if it is at a low MW to get the same Rayleigh ratio for it
What equation is used when the molecules is 1/10th of the wavelength
What about when the solutions are not idea
Diff equation
When not ideal have to add in even another term
On sheet
Explain how to do the practice question of finding Rg and MW for an ideal solution
Use the ideal 1/10th equation (on sheet)
Have to make a linear curve of 1/Rtheta vs sin^2 (theta/2) /Rtheta
Start by dividing R theta and MW K c on either side
Multiply 1/R theta into the brackets
Then get form of y=mx + b
Plug in values for each R theta and theta
Plot values on graph, get slope and y int from linear equation on graph
1/ MW K c = slope, isolate for MW (kg/mol, make sure to account for powers)
In the same way, use the slope value to isolate for Rg (nm)
On sheet
What is multiangle light scattering
A type of static light scattering
Multiple detectors measuring the light scattering from the protien at the same time
The system is coupled to something that separates the protien based on size , like SEC, field flow fractionation, asymmetric field flow fractionation
Can be used to see if complexes were made and for quality control
What is dynamic light scattering (DLS)
It’s measured as intensity vs time
Also called photon correlation spectroscopy because seeing correlation between photons that are scattered
When molecules move around in solution, they sometimes scatter in phase (higher intensity) or out of phase (lower intensity)
The changing in intensity depends on the diffusion coefficient of the solution and the molecular size
What is actually measured in DLS
What is the size range
How is the data displayed
The hydrodynamic diameter or stokes radius (Rh)
So not the bare protien diameter, it’s the protien with the water molecules that move with it
1-5000 nm
Shown as a histogram with fraction/percentage vs hydrodynamic radius (Rh)
What happens if larger diffusion coefficient in DLS
Faster diffusion, smaller particle
If smaller, slower diffusion , larger particle
Equations in DLS
On sheet
What are the uses of DLS
Main advantage
Measur particle size and distribution
Can find small changes in mean diameter
The data gives info on the samples characteristics like aggregation (if aggregating, gain larger hydrodynamic radius, one peak on histogram)
Main advantage: size info can be found in minutes (fast)
Advantages of DLS
Non-invasive (no labelling needed)
Quick and doesn’t need standards
Small amounts (microlitres)
Sensitive (doesn’t need high concentrations of sample)
Good for trace amounts of aggregate (larger molecules show up well)
