Unit 10 Flashcards
describe electrophoresis
separation technique based on the differential rates of migration of charged species in an electric field
what molecules moves towards the cathode?
pos charged molecules move towards the cathode (neg electrode)
what molecules moves towards the anode?
neg charged molecules move towards the anode (pos electrode)
what does the direction and rate of migration depend on?
physical characteristics of the electrophoresis system and solute molecules
what are 2 most important aspects of the solute molecules electrophoresis?
their size and charge
eg. larger molecules don’t diffuse through as well
electrophoresis can be carried out in what two modes?
- free solution (eg. capillary zone electrophoresis: CZE)
2. with the aid of a stabilizing or supporting medium (eg. paper, gel, etc…)
what is the role of solvent reservoirs?
buffers to control pH of the system
what kind of capillaries are used for capillary electrophoresis?
describe the properties
narrow bore capillaries
- made of fused or modified silica
- diameter: 10-200 um
- range of potentials: 5-30kV (pos or neg)
what detectors are used by capillary electrophoresis?
same as HPLC detectors
- absorbance
- fluoresence
- mass spectrometry
- conductivity
what happens when a charged particle is placed in an electric field
it will be accelerated and start to move towards the electrode with an opposite charge
what is the electrophoretic velocity?
the rate of migration of the charged particle through an electric field
what is the unit for electrophoretic mobility?
cm^2/V*2
what is the unit for the applied field?
V/cm
what is Mep?
charge density
what are factors affecting mobility of a charged analyte?
- strength of applied electric field
- temp of the separation system
- pH of the separation buffer
- type of buffer ions and ionic strength of the separation buffer
- size and shape of analyte molecules
- charge of the analyte molecules
what happens when a high voltage is applied across the fused-silica capillary with a buffer solution?
what is this called?
the buffer liquid starts to migrate towards the cathode
the rate of this migration is the elctroosmotic flow velocity
what is Veo?
electroosmotic flow velocity
what is the unit of Veo?
unit * cm/s
what causes the elctroosmotic flow velocity?
the charge distribution at the silica/buffer interface and the creation of an electrical double layer
when a voltage is applied, cations in the diffuse portion of the double layer migrate in the direction of the cathode, carrying water with them
when is the elctroosmotic flow more pronounced? why?
at high pH
there are more O- at the surface
what is the equation for electroosmotic flow velocity?
Veo = Meo x E
what is the direction of voltage of electroosmotic flow?
anode to cathode
what are common buffers for capillary electrophoresis?
when is each of them used?
phosphate
acetate
borate
they are all used for different pH ranges
what is Vtot?
- the sum of electrophoretic velocity (Vep) of an ion and the electroosmotic velocity (Veo)
Vtot = Vep + Veo
OR
2. Vtot = L/Tm
L = length of column from inlet to detector
Tm = total migration time
how does the increase of electroosmotic flow affect the separation time?
reduces separation time
at moderate pH values (pH >3), which is higher?
a) Meo
b) Mep
what is the result of this?
Meo > Mep
causes anions to migrate towards the cathode where the detector is
at low pH values, describe the net migration.
Meo is weak and anions may never reach the detector unless the polarity of the instrument is reversed in order to change the location of the detector from the cathode to anode end
what is an example of a small organic and inorganic ion?
malic acid
what is an example of a small amino compound?
putrescine
what is an example of a phenol, polyphenol or pigment?
chlorogenic acid
negative charges of amino acids come from what functional groups?
carboxylic groups
positive charges of amino acids come from what functional groups?
amine
what is IEP?
describe it
isoelectric point
the pH at which net charge is zero
at high pH, the net charge on proteins is…
a) more positive
b) more negative
higher pH = more negative
