LECTURE 8 - Capillary electrophoresis

Elaborate on capillary electrophoresis

In this technique components of a solution are separated by applying a voltage of 30 kV from end to en of a fused silica capillary tube

Explain the process

1. sample injection: done by capillary imersion in a sample with either the application of pressure or an electric field. Sample start moving fast in tube due to electric field

2. Then when the sample reached the buffer it slows down a bit

3. When the slow down occurs the ions pile up in narrow band (stacking)

4. Then ions will have different mobilities and migrate through the tubes at different speeds. They can seperate based on the charge to size ratio.

Describe the inside of a capillary tube

The tube is a glass tube covered in silanol grps and then there is the electric double layer which contains the diffuse bulk layer and then the bulk. Essential how it works is the tube will be negative due to the silanol groups and then the positive charges of the buffer will be attracted to these negative charges creating a sort of cloud of positive charges surrounding the tube and whenan electric field is applied they will all move towards the cathode which in turn moves the bulk solution as well. so even if the analyte in question is negative it could potentially be brough to the cathod depending on pH of buffer.

Why is this technique attributed to better resolution

As it has a very small width of solution chamber

What is stacking

It is when the analyte is foccused into a narrow band within the capillary. When focussed like this it results are resolved better

In an electric field cation are attracted to what

The cathode

In an electric field anions are attracted to what

the anode

what they term pump in electrophoresis is what essentially

It is the more common name to electroosmosis in which the extra cations in the diffuse layer result in net momentum towards the cathode. amount of positive ions in diffuse layer is due to the pH

Describe electroosmosis velocity

It is the uniform mouvment of susbstance inside the capillary away from the anode and towards the cathode

Describe the hydrodynamic velocity

It is the non uniform. mouvement of substance in capillary towards an area of high pressure to low pressure

define electroosmotic flow

ions in electric double layer are the pump that drives the electroosmotic velocity profile

define hydrodynamic flow

the mouvment of ions is driven by the pressure difference

If you want to measure the electroosmotic velocity what do you do

To measure it you need to add a neutral molecule to the sample and see how it responds, This is important as it will only be influenced by the diffuse bulk layer and not its charge

What is the relationship between electroosmotic mobility and the surface charge density on silica

It s proportional

What is the relationship between the electroosmotic mobility and the ionic strength of the background electrolyte

inversely proportional to the square root of the ionic strength

define the appparent mobility

It is the mobility we see like how is it actually moving in the capillary

What would the apparent mobility of an cation analyte be

The apparent mobility would be greater than the electrophoresis mobility as Mep and Meo would both be positive

What would be the apparent mobility of a anion analyte

Apparent mobility would be smaller than the electrophoretic mobility since the Mep and Meo would have different signs

Elaborate on the role of pH in capillary electrophoresis

When pH is high the silanol groups are highly charged and are deprotonated so electroosmosis will be more powerful and anions can be brought to the surface as the diffuse layer will be larger. When pH is low however, the OH will protonate the silanol group surface, resulting in neutral groups and therefore since its neutral less positive charges will be attracted to it resulting in a thinner diffuse layer where the pump of ions will be slower and anions may never reach the detector

What is sieving

This is a technique where the capilaries are filled with a meterial like a gel that allows the small molecules to pass more freely than larger molecules.

what are the applications of CE

1. DNA sequencing

2. Forensic analysis of protein, DNA, explosive, guynshots, ink and drugs

3. Drug analysis

4. Analysis of starches and polysaccharides

What is flow reversal

this is when there is a charge reversal which is created when you add an ampipathic molecule to the surface which changes the surface in the capillary to positive. The diffuse layet in this case will be composed of excess anions taht will pump ions through as they move to the anode and the bulk rest of the ions will also move that way as well.

When you calculate the moles injected what is important to be considered

That the concentration should be in mol/m3, meaning you need to divide by 1000

What is so special about the detectors in capillary

They are made in a way to increase the pathlength resulting in higher absorbance values this could be either the bubble method or just a 90 degree angle path change

What is indirect detection

In indirect detection we add a substance within the mixture of our tested sample that we know absorbs at a certain wavelength as a background molecule. As the target molecules pass the detector there will be a recorded drop of absorbance with that drop it is evident that a target analyte has base by