July 22nd Flashcards
electrophoresis
separates DNA and proteins based on size
UV spectroscopy
passing UV light through a chemical species and plotting wavelength vs absorbance - useful for studying double bonds or atoms wiht lone pairs bc need electrons that are free to interact
H-NMR
type of nuclear magnetic resonance
- type of proton= how many peaks
- desheilded proton = to the left ( means that there are lots of electron withdrawing groups)
- splitting = hydrogens on adjacents carbons will split a peak into n + 1 subpeaks, n = # of hydrogens on adjacent carbon
H-NMR
type of nuclear magnetic resonance
- type of proton= how many peaks
- desheilded proton = to the left ( means that there are lots of electron withdrawing groups)
- splitting = hydrogens on adjacents carbons will split a peak into n + 1 subpeaks, n = # of hydrogens on adjacent carbon
Electrophoresis
separate proteins - charge and size
electrolysis cell so anode is positive ( neg proteins travel here)
polyacrylamide gel (PAGE) is standard gel used ( acts like a sieve- allowing smaller ones to pass faster)
PAGE limitations
mass and charge ( so it’s good if say proteins have similar size)
SDS-PAGE
separates proteins based on their size only - they all get same negative charge
isoelectric focusing
separate proteins based on PI point
- acidic gel at positive anode
- electrolytic cell as well so anode is positively charged (acidic amino acids will be attracted to the positive side- this is also where low pH is) they will eventually stop when they reach their PI
what pH is at the anode in isoelectric focusing
low pH and positive anode
thin-layer chromatography
spots on the stationary phase, mobile phase runs through it
pare is highly polar
paper = polar (silica paper) and the mobile phase is polar
thin-layer chromatography
spots on the stationary phase, mobile phase runs through it
is silica polar
very
column chromatography
column filled with silica or alumina beads as absorbent (stationary phase) and solvent passed through
- first eluted = less polar
what travels highest in TLC
nonpolar substances ( bc paper is silica)
RP-chromatography
-N-M-N, so if RP than the mobile phase is polar
size -exclusion
beads contain tiny holes, so the biggest ones get eluted first
HPLC
N-M-N, the mobile phase is non-polar
Hills coefficent
numerical value for cooperativity in enzyme kinetics
- the value of hills constant indicates the nature of binding by the molecule
- if hills is greater than 1 = positive cooperative binding
- if hills less than 1 = negative cooperativity - ligand binds, the affinity for further ligans decreases
- hills= 1 = no cooperativity
cooperativity in enzymes
- show a sigmondal (S-shaped) graph- due to cooperativity amoung substrate binding sites
- 2 states; high affinity (R) relaxed state, or (T) low-affinity tight state
- binding of substrate, encourages other enzymes to go to R states
- hills coeficient tells us about cooperativity ( if >1 = positive, if <1 = negative
catalytic effecicency
kcat/km ( high efficentcy if large kcat or small km= high affinity)
kcat
measures the number of substrates turned ober/ enzyme/second
extractions
2 immiscible solvents - form 2 layers
- seperatory funnel –> denser one on bottom ( commonly the aqueous layer is more dense)
are dipole-dipole interactions in molecules going to put them in the aquous layer
not commonly no
simple distillation
- differences in BP - lower will vaporate first
- below 150 and have atleast 25 C difference between them
vaccum distillation
if BP is over 150 - using a vacuum lowers the ambient pressure and thereby decreasing the heat needed to boil a liquid
fractional distillation
to separate 2 with simular BP (less than 25 degrees)
- fractional column ( squiggly) - surface area is increased, as vapor rises it condenses on this surfaces and refluxes down untill it vaporizes again
is cellulose polar or nonpolar
polar (used as stationary phase on TLC) - hella OH
gas chromatography
same principle as column chromatography but the elutent is the gas, how well do they adhere to the absorbant
- and on the edges of column is crushed metal or polymer in a coil which is heated - particles that adhere to the sides - eluted last
- smaller ones travel faster
- injected compound must be volatile
just plain old simple column chromatography
N-M-N
mobile phase is the solvent and the column is filled with silica or alumina beads
mass spectrometry
ionization and fragmentation of compounds, fragments run through magnetic feild, which seperates mass-to-charge ration
- can determine total mass or relative concentrations of different fragments
MRI
magnetic resonance imaging - most actually measure H-NMR spectra of water molecules in different environments in the body
spectroscopy
measures energy differences by determining the frequencies of electromagnetic radiation absorbed by the molecules
The basic principle shared by all spectroscopic techniques is to shine a beam of electromagnetic radiation onto a sample, and observe how it responds to such a stimulus. The response is usually recorded as a function of radiation wavelength
spectroscopy is the study of the interaction between matter and electromagnetic radiation (via electron spectroscopy, atomic spectroscopy, etc). Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, by a prism.
IR vs UV spectroscopy
- the wavelength of infrared light is longer than uv/vis. -
- Infrared absorption by molecules corresponds to differences in vibration energy. Infrared spectroscopy can therefore be used to identify molecular vibrations and uniquely recognize compounds and functional groups
- Uv/vis absorption by molecules correspond to differences in the (covalent) bonding of atoms.
- used for studying lone pairs or molecules with pi bonds such as transition metal ions, highly conjugated organic compounds, and biological macromolecules.
UV spec
- wavelength of max absorbance can be determined
- record absorbance and plot against wavelength
- absorbance caused by electronic transitions between orbitals
- works bc molecules with pi electrons or non-bonded electrons can be excited by UV light to higher antibonding orbitals
NMR
certain atoms have atomic nuclei that have magnetic moments
when they are placed in a mag feild, their moments tend to aligh with or against the direction of applied field
if nuclei align with applied field…
they are said to be in alpha state= lower energy
