MAAP: Capillary Zone Electrophoresis Flashcards
Describe how the CZE instrumentation works
A capillary (10-25cm long) of diameter < 1mm is filled with a ‘running buffer’ which is an electrolyte containing an aqueous buffer at a specific pH usually between 3-9.
One end of the capillary is in the ‘destination vial’ (filled with running buffer and an electrode attached to a high voltage supply) and the other in either
- the ‘source vial’ (also filled with running buffer and an electrode attached to a high voltage supply) or a
- sample vial
Once the sample has been introduced onto the capillary via the sample vial the capillary is returned to the source vial and a potential difference (Voltage) is applied.
This results in a current to flow usually from the anode (at the source) to the cathode (at the destination) in which charged analytes migrate and pass through a detector.
The plot of detector response against time is known as an electropherogram.
How do analytes move through the capillary to reach the detector?
Analytes move based on charge and size
Although negative analytes have a desire to stay at the positvely charged anode end, the EOF is stronger than this so forces them to the cathode
Describe EOF
The capillary is made of fused silica thus will have Si-OH at the inner surface.
If the running buffer (in the capillary) is above pH 3 (i.e. presence of some HO- ions) then some of the Si-OH will be de-protonated to SiO- (H+ + HO- “ H2O)
Si-OH + -OH → Si O- + H2O
The SiO- groups then attract positively charged cations (e.g. Na+) from the running buffer.
These form a fixed layer at the inner surface of the capillary and are held tightly through non-covalent interactions.
However small hydrogen ions have been replaced by much larger cations from the buffer.
Thus all the created Si-O- cannot be neutralised because there is not enough room to fit enough the huge cations at the surface.
The fixed layer will still have an overall negative charge.
Thus a second layer of cations from the running buffer, forms on top of the inner fixed layer.
Since this layer is further away from the Si-O- groups it is less tightly held and because of this is termed the mobile layer.
When an electrical field is applied, the cations in the mobile layer will start to move towards the cathode.
The mobile layer of cations is solvated in the buffer solution, that is, they are surrounded by water molecules, thus will drag the bulk buffer solution along, causing the EOF.
Because of the bulk buffer solution movement towards the cathode, neutral and charged analytes will also progress towards the cathode and therefore pass through the detector
Why is temperature control of the capillary important?
How is it done?
It is important to regulate the temperature of the environment around the capillary to ensure consistent separations. (Ensures consistant peak shape and Retention time)
To achieve this, capillaries are often inserted into cartridges which are placed in the CE instrument.
Temperature controlled air or liquid coolant is then forced through the cartridge to regulate the temperature.
What are methods of sample introcution onto the capillary?
What detectors can be used?
What ways can seperation of analytes be manipulated
- pH change
- Buffer conc change
Describe changing pH effects on manipulation of seperation
Describe changing buffer conc effects on manipulation of seperation
What is the main advantage of CZE?
Flow Profile
In a pressure driven system, like HPLC, frictional forces at the liquid-solid interfaces result in a substantial pressure drop.
These liquid-solid interfaces occur all through the system at the walls of the tubing and between the mobile phase and the packing.
As a consequence of this parabolic flow (see Figure (b) next slide, the velocity of the mobile phase is highest in the centre of the tube and nearly zero next to the walls. This velocity gradient results in band broadening)
In electrically driven systems (such as CE), the driving force of the EOF is uniformly distributed along the entire length of the capillary.
As a result there is no pressure drop and the flow velocity is uniform across the tubing diameter, except very close to the wall where again the velocity approaches zero.
However this area is insignificant compared to the rest of the flow, see Figure (a) above.
Higher modes
Capillary Gel Electrophoresis (DNA analysis)
Micellar ElectroKinetic Capillary Electrophoresis (for neutrals)
Iso Electric Focusing (for ampholytes)
Capillary Electro Chromatography (Hybrid of HPLC and CE)
