Question

In open tube CE a) How does chiral analysis work and with what additives? b) explain with detail why use coated capillaries? c) explain with detail why open tube CE shows high resolution towards certain samples? d) what are the differences between electrophoresis and electroosmosis in detail?

Answer 1

a) Chiral CE separation is achieved by adding a chiral selector to the BGE. The enantiomers form fast, reversible equilibria with the selector. This means that part of the time an enantiomer migrates free in the BGE, and part of the time it migrates as an enantiomer-selector complex. The apparent mobilities of the enantiomers will change depending on the strength of the complex (i.e. the equilibrium constants) and the mobility of the enantiomer-selector complex. In order to separate the enantiomers, a few requirements need to be met. First of all, either the enantiomers or the selector have to be charged. Furthermore, the enantiomers of a chiral molecule need to have different affinities for the chiral selector, and/or the enantiomer-selector complexes need to differ in mobilities. . In the bottom trace, 10 mM dimethyl-β-cyclodextrin is added as the chiral selector to the BGE and the enantiomers of all LAs are efficient.

Cyclodextrins (CDs) are commonly used as chiral selectors, as there is a wide variety of CDs available and they absorb little UV light at the common detection ranges. This makes CDs rather popular, although the disadvantages are that they are poorly characterised and only exist in one absolute configuration. The latter means that one cannot change the order of migration of enantiomers in a system by exchanging the chiral selector from one configuration to its mirror image. The poor characterisation can give supplier or batch-to-batch reproducibility issues. Other chiral selectors that have been used are e.g., chiral crown ethers, optically active micelles, bile salts, macrocyclic antibiotics, proteins and ion-pair selectors. Method Development In CE, the analyte needs to carry charge, either by itself or by complexation with e.g. a chiral selector. In chiral CE, different starting points are applied for the enantiomeric separation of basic, acidic or neutral analytes. The strategy to select a starting point for a basic analyte is usually relatively simple. At a low pH, at least two pH units below a basic analyte’s pKa, it is mainly (≥ 99%) charged and at a low pH (i.e. below pH 4) also the electro-osmotic flow (EOF) is low. This means, that either a charged or an uncharged chiral selectors can be used. The advantage of using an uncharged selector is that it does not contribute to the ionic strength of the electrolyte and therefore does not increase the current and Joule heating. An uncharged selector migrates with the electro-osmotic flow and the separation window for the enantiomers is between the migration of the unaffected analyte, i.e. the migration velocity of the enantiomeric analytes with no selector present. For basic analytes with a BGE at a low pH and slow EOF, the separation window is rather large. the advantage of a negatively charged selector is that it migrates slower than or in reverse direction of the EOF, which enlarges the separation window further. The disadvantage of a charged selector is that it can contribute considerately to the ionic strength of the system, resulting in (often too) high currents.

b)Capillary coating. The reduction of or elimination of EOF can be useful to enable direct electrophoretic separations to be performed. More compelling is the ability to eliminate solute adsorption. A variety of coatings is possible including some phases used for capillary gas chromatography. The use of hydrophilic coatings can be useful in suppressing adsorption of hydrophobic compounds. Electrostatic binding can also be suppressed. Hydrophobic coatings, in conjunction with nonionic surfactants as buffer additives, appear promising as well. Many companies are beginning to introduce bonded-phase capillaries. These capillaries should further extend the range of compounds applicable to separation byCapillary zone electrophoresis ( CZE).CZE is very useful for the separation of proteins and peptides since complete resolution can often be obtained for analytes differing by only one amino acid substituent. This is particularly important in tryptic mapping where mutations and post-translational modifications must be detected.

New coating procedures that prevent molecular adsorption to the capillary walls during CE separations continue to be attractive due to the simplicity of coating preparation, use, and regeneration. Several relevant coatings procedures that do not rely on covalent modification of the walls, but use surfactants, polymeric materials, or nanoparticles.

d)differences between electrophoresis and electroosmosis

What is Electrophoresis?

Electrophoresis is a technique of separating molecules based on their sizes. Fundamental for this separation is the charge of the molecule and their ability to move in an electric field. This is the most common and main technique in molecular biology to separate molecules, especially DNA and proteins. This is mostly in use because it is relatively easy and inexpensive. The apparatus for the electrophoresis can be bit complicated, and preparation of it takes some time. But we can easily make an electrophoresis apparatus from the things we have in the laboratory. Electrophoresis techniques can vary depending on our purposes. We can use one dimensional electrophoresis for the separation of DNA or protein. Two dimensional electrophoresis is used when more resolved samples are required (as in the case of finger printing). A gel is used as the support medium to separate the molecules. This gel can be prepared as flat sheets or in tubes. Basis of this procedure is to separate molecules depending on their rate of movement through a gel when an electric field is supplied. Negatively charged molecules like DNA tend to travel towards the positive pole in this electric field while positively charged molecules tend to travel to the negative pole. Two types of gels are used in electrophoresis as agarose and polyacrylamide. These two have different resolving powers. The gel acts as a sieve to filter the different sizes of molecules. The electrostatic charges set up in the gel act as the force.

Separation depends upon the mobility of the ions.

F=fv=ZeE

V=ZeE/ f

F= force acting on a particle

f=frictional coefficient

V= average migrating velocity

Z= charge of the migrating particle

e=elementary charge

E= strength of the electric field

The necessary conditions for the electrophoresis are relatively simple. When making the gel and running the sample, a buffer is used. Markers and dyes are used for the visualization purposes.

What is Electro-osmosis?

This is the process of moving a liquid through a material using an applied electric field. The movement can be through a porous material, along a capillary, membrane etc. This can be used as a separation technique (especially capillary electro-osmosis). The velocity of the liquid is linearly proportional to the applied electric field. It is also dependent on the material used to build the channel and the solution used. In the interface, solution and material have obtained opposite charges and, this is known as an electrical double layer. When an electrical field is applied to the solution, the electrical double layer moves by the resulting Coulomb force. This is known as the electro-osmotic flow.