fundamental techniques Flashcards
light scattering
aka photocorrelation spectroscopy
This method can determine size distribution of particles in a suspension
If the particles aren’t round also need to consider rotational Brownian motion effects on scattering
xray diffraction
one application in crystallography
need to use braggs law n*lambda = 2d sin(theta)
to related xray diffraction pattern to 3d locations of particles
need less than 1.5A for good results but can get up to 1A resolution
neutron diffraction
can get up to 2A resolution
used to identify location of hydrogens and waters.
Can swap H and deuterium to identify solvent accesibility and macromolecular dynamics
problem is requires large beams or long exposure
Complementary to both NMR and xray diffraction
multiphoton microscopy
used for imaging living cells from molecular to organismal level
Can image deeper since it uses near ifrared lasers
confocal microscopy
a very narrow beam of light to penetrate the sample at different levels
Can build 3D models
both live and dead cells
scanning electron microsocpe
detects surface topology.
Similar to TEM but only surface topology
diffracion limit
resolution =(wavelength)/(numerical aperature size)
For optics today its about
Resolution=lambda/2.8
atomic force microscopy
resolution in angstroms
can resolve the topology
or do pulling
diffracion limit
resolution =(wavelength)/(numerical aperature size)
optical limit abuot 1 microns
sedimentation experimenets
can identify mass and molecular weight of sample
detect amount of aggregates
determining size of proteins native state (monomer vs trimer, vs dimer etc)
determining shape of molecule
detect change in conformation of protein
study formation and stoichiometry of complexes (receptor-ligand or antigen-antibody)
types of absorption spectroscopy
*xray : excitation of inner shell
*uv-vis
IR
microwave
radiowave (electron spin resonance and NMR)
Fraunhofer lines
absoprtion spectra lines each one representing an element (although some represent the lines in the hydrogen atom Balmer series)
Balmer series
spectral lines of the hydrogen atom (4 in visible spectrum, 2 in UV)
- for tranisition from n>2 to n=2 orbital
Energy difference between orbitals
E-E0= -Rhc*(1/n2^2 - 1/n1^2)
Lyman series
lines for transmission from n>1 to n=1 orbital
xray absoprtion spectroscopy
determines local geometric and electronic structure
UV VIS absorption spectroscopy
detemrine chemical kinetics
IR absorption spectroscopy
vibrational spectroscopy
IR is absorbed at resonant frequencies
measures changes in dipole moment
microwave spectroscopy
rotational spectrosopy
microwave spectroscopy
rotational spectrosopy
measures how dipole interacts with EM field
microwaves cause changes between 5 rotational modes
diatomic, linear, spherical, asymmetric tops, and symmetric tops
electron spin resonance spectroscopy
unpaired electrons are measured
detects transitions in a magnetic field
spins splitting based on rules
can probe structural properties such as those for Vimentin
Nuclear magnetic resonance
changes to nuclei in magnetic field
can determine structure and dynamcis of proteins and nucleic acids
fluorimetry
You can attach a flurophore to a species or part of a cell
Then you shine light to excite it and cause it to fluresce, From there you track what is happening in time and space
tryptophan fluorescence in proteins too!
can track how proteins are folding
Raman spectroscopy
uses monochromatic light
identifies molecules, chemical bonds, and intramolecular bonds
Also study substarte to an enzyme
used to confirmed low frequency phonons in proteins and DNA
