Capillary Electrophoresis Flashcards
what is electrophoresis?
separation technique based on different rates of migration of charged species in E field
cations move toward cathode, anions move towards anode
In what direction do cations and anions move in electrophoresis?
cations move toward cathode, anions move towards anode
The cathode has a ____ charge. The anode has a ____ charge.
cathode = negative anode = positive
Migration of molecules in the E field will depend upon factors relating to (2):
physical characteristics of electrophoresis system
solute molecule characteristics (size, charge)
migration describes ____ and ____ of the molecule’s movement
direction
rate
describe the general design/features of a capillary electrophoresis system
2 separate solvent reservoirs, connected to high-voltage power supply (1 is anode, 1 is cathode)
capillary runs between them, with detector in middle (collect data on molecule movement between reservoirs)
What type of capillary is used for CE? What other detectors can this be used with?
narrow bore capillaries
made of fused silica (or modified silica)
absorbance, fluorescence, MS, conductivity…
When a particle is placed in an E field, what happens?
accelerates towards electrode with opposite charge
What is ‘Vep?’
electrophoretic velocity (cm/s) (rate of migration of particle in E field)
What does ‘Uep’ stand for
electrophoretic mobility (cm/sV)
The electrophoretic mobility is proportional to:
the charge density (q/rs)
What factors affect mobility of a charged analyte in CE? (6)
strength of E field temperature pH of separation buffer buffer ion type/ionic strength size/shape of analyte molecules charge of analyte molecules
What happens to the buffer liquid in CE system when a high voltage is applied across the fused silica capillary? What is this called?
buffer liquid starts to migrate towards cathode
Veo; electroosmotic flow velocity
T/F: electroosmotic flow velocity and electrophoretic velocity have the same unit
true
What causes the electroosmotic flow velocity?
charge distribution at silica/buffer interface + creation of electric ‘double layer’ (fixed layer + mobility/diffuse layer)
apply voltage -> cations in mobility layer migrate to cathode, carries water with them
The electroosmotic flow velocity effect is more pronounced at _____. Why?
high pH
many O- charge at surface
What causes the formation of the electric double layer in the capillary?
negative charged capillary walls attract cations (+ charges) from the buffer
some bind tightly -> fixed layer (but doesn’t fully neutralize the neg. charge)
some cations are still attracted towards the walls -> diffuse layer
What is the net charge within a CE capillary?
neutral (cancels out)
What are some common buffers for CE and their pH range?
phosphate (1.14-3.14)
acetate (3.76-5.76)
phosphate (6.2-8.2)
borate (8.14-10.14)
What CE buffer can be used at pH 9?
borate
pH range 8.14 - 10.14
What CE buffer can be used at the lowest pH?
phosphate
pH 1.14-3.14
What CE buffer is useful at a neutral pH?
phosphate
6.2 -8.2
What are some zwitterionic buffers for CE?
MES (5.15-7.15) PIPES (5.8-7.8) HEPES (6.55-8.55) tricine (7.15-9.15) Tris (7.3-9.3)
What is the ‘total velocity’ of the analyte in CE?
Vtot
sum of electrophoretic velocity (Vep) + electroosmotic velocity (Veo)
Vtot can be expressed as: (3)
Vep + Veo
(Uep + Ueo) x E
L/tm (total migration time)
how does electroosmotic flow affect separation time?
increasing Veo -> decrease separation time
What do Utot and Ueo values in CE correspond to in chromatography?
Utot = Tr (total retention time) Ueo = Tm (dead time)
Utot-Ueo corresponds to adjusted retention time (Tr-Tm)
In cations, Uep and Ueo flow in ___ directions, while in anions the flow in _____ directions
identical directions (toward cathode)
opposite (Uep flows toward anode; negative flow)
What has ‘negative flow’ in CE?
anions electrophoretic flow
How can anions still be detected, if the electrophoretic flow direction is away from the detector?
at pH>3, Ueo (electroosmotic flow) is higher than Uep; so overall NET MIGRATION will be towards cathode
*or; reverse polarity of instrument (detector at anode instead)
What happens to anions if the pH is low (<3)?
Ueo is weaker than Uep; so anions will never reach detector (unless polarity is reversed)
What food chemicals can be separated with CE?
small organic/inorganic ions small amino compounds (AA, biogenic amines, heterocyclic amines) peptides/proteins phenols/polyphenols/pigment carbs vitamins pesticides antibiotics
What detector types are used with CE?
Spectrophotometry (absorption, fluorescence, thermal lens, raman, chemiluminescence)
MS
EC (potentiometry, amperometry)
what allows for the separation of AA in CE?
charges on functional groups (positive, negative, etc)
AA functional groups with neg charges: ______
AA functional groups with + charges: ______
neg: carboxylic func. groups (ex: glutamic acid)
positive: amine func. groups (ex: arginine)
As pH increases, what happens to the net charge on proteins?
decreases (change from + to negative)
When the protein net charge is neutral, this is called the ______
isoelectric point (specific pH)
