Analytic Techniques Flashcards
Extraction in protein purification
have to lyse cell membranes to remove proteins
What do you treat protein mixtures with during extraction?
protease inhibitors
Where are proteins in centrifugation?
lighter proteins stay suspended in the supernatant
heavier cell debris and such moves to the bottom
How can you use salt to separate proteins?
initially the salt will increase solubility of the protein by breaking apart side chain reactions
then, when enough salt is added it will compete with the protein for solvent to be broken down and the protein will precipitate out
use the optimal amount of salt that the protein precipitates out
Thin layer chromatography
use polar silica as stationary phase
separate compounds on polarity
use Rf ratio
What type of molecules move the furthest in thin layer chromatography?
nonpolar substances
interact poorly with the polar stationary phase
Rf
ratio of how far a substance traveled in TLC
has to be between 0-1
Iodine in TLC
helps us visualize the compounds
High performance liquid chromatography
uses a polar stationary phase
polar materials have a longer retention time
Reverse phase HPLC
uses a nonpolar stationary phase
Gas chromatography
mobile phase is gas and stationary phase is liquid
compounds with low boiling points evaporate first
Size exclusion chromatography
captures small molecules in gel
Anion-exchange chromatography
has positive beads to attract anions
Cation-exchange chromatography
has negative beads to attract cations
Salt buffer in ion-exchange chromatography
releases attracted molecules
Affinity chromatography
ligand binds to a specific receptor in the column
Electrophoresis anode and cathode
anode is positive
cathode is negative
SDS limits
does not break down disulfide bridges
need a reducing agent to break down disulfide bridges
What travels the furthest in SDS page?
smaller molecules
Isoelectric focusing
a type of electrophoresis where molecules will end up at pH that equals there pI
Immunoassays
rely on antibodies for specificity/identification
Example of an immunoassays
Western blotting
ELISA
antigens marked with fluorescent labels bind the molecule of interest can use spectroscopy to see how many molecules binded
Beer-Lambert Law
A = ecl
At what wavelength do proteins have max absorbance? Why?
in the UV spectrum
as aromatic/conjugated pi bonds
Extraction
separate liquid mixtures into organic and aqueous phases
Organic phase in extraction
non-polar
Aqueous phase in extraction
polar
Where are uncharged acid and bases found in extraction?
in the non-polar organic phase
Distillation
separates liquids by their boiling point
Fractional distillation
can separate liquids with similar boiling points
Vacuum distillation
lowers atmospheric pressure to allow high boiling points to evaporate
Recrystallization
recrystallize a solid precipitate to filter out impurities that may have been in the original precipitate
Nucleation
initiates the recrystallization process
Spectroscopy
analyzes molecules based on interaction with electromagnetic radiation
How does IR radiation work?
makes polar bonds bend and stretch
Where is O-H found in IR?
a broad peak at 3100-3500
Where is C-H found in IR?
a stretch at 3000
Where is C=O found in IR?
a sharp peak at 1650-1780
UV-Vis Spectroscopy
looks at movement of electrons
good for determining conjugated pi systems
How can you find the color of a compound from UV-VIs spectroscopy?
colors that you don’t absorb give the color of the conjugated system
What is UV-Vis spectroscopy good at measuring?
conjugated pi systems / aromatic compounds
Where are less shielded protons found in NMR?
downfield to the right
What makes a proton less shielded in NMR?
the presence of electronegative species that remove ED
How many number of signals are found in NMR?
number of sets of equivalent hydrogens / carbons
Where is the position of signals in NMR?
look at shielding to shift upstream or downstream
What does the size of the signal in NMR tell us?
more protons means more area/larger size
How is splitting in NMR determined?
n+1, where n is the number of adjacent hydrogens
Is there splitting in 13C NMR?
no
Mass spectroscopy steps
1) Ionize by shooting electrons at molecule
2) Accelerate in E-field
3) Curve in B-field to tell us the mass
Parent ion in mass spectroscopy
Generated by the loss of only on electron
Is the peak the furthest to the right on graph
What can the parent ion tell us in mass spectroscopy?
the molecular weight of the molecule
Parent ion abbreviation
M+
