Charge silica capillary wall

In the pH range 4—7, small pH changes have large effects on the charge of the fused silica capillary wall, and thus the EOF. If a BGE pH in this range is needed, coated capillaries (permanently or dynamically) should be considered. Also, more robust systems are obtained if the BGE consists of both a buffering co-ion and a buffering counterion. ... 

Polycations are strongly retained by the bare silica capillary wall, since they can ion-pair at multiple sites on the anionic wall. Since all electrostatic bonds must be broken to free the solute, this problem becomes increasingly severe as the number of positive charges increases. Polycations may include small molecules such as tetrazoles and even small peptides. Either permanently coated or charged-reversed capillaries are usually required to separate polyvalent cations. Monovalent cations show some wall effects, but these do not preclude the bare silica capillary from being used. Polyanions and monovalent anions can usually be separated on bare silica. 

The mechanism by which analytes are transported in a non-discriminate manner (i.e. via bulk flow) in an electrophoresis capillary is termed electroosmosis. Eigure 9.1 depicts the inside of a fused silica capillary and illustrates the source that supports electroosmotic flow. Adjacent to the negatively charged capillary wall are specifically adsorbed counterions, which make up the fairly immobile Stern layer. The excess ions just outside the Stern layer form the diffuse layer, which is mobile under the influence of an electric field. The substantial frictional forces between molecules in solution allow for the movement of the diffuse layer to pull the bulk... 

Sample adsorption to the silica wall is a problem in HPCE, one that is highly undesirable. As we mentioned earlier, adsorption can be minimized by proper buffer selection, additives, or chemical modification of the surface. The selection of pH is one of the simplest separation parameters to manipulate and is critical to the success of all electrophoretic separations. The pH of the media will determine the charge of the sample and the charge of the silica surface. At low values of pH, the capillary wall is protonated, the EOF... 

Fig. 17.2. The distribution of charges at the internal wall of a silica capillary. x is the length in cm from the center of charge of the negative wall to a defined distance, 1 = the capillary wall, 2 = the Stern layer or the inner Helmholtz plane, 3 = the outer Helmholtz plane, 4 = the diffuse layer and 5 = the bulk charge distribution within the capillary.

A double layer of charged counterions is formed adjacent to the wall. In the case of fused silica capillaries a fraction of the cations in the double layer adsorb strongly to the wall,... 

In capillary electrophoresis instruments, the electro-osmotic flow is used to impose, on all charged species in the sample, a direction of migration that is oriented from the anode towards the cathode. An increase in the electro-osmotic flow vEOS decreases, at the detector, the gap in migration times of ions travelling in the same direction. The use of fused silica capillaries partially deactivated by coating the inner wall allows modulation of the electro-osmotic flow. A voltage gradient can also be used to this end. 

The inside capillary wall controls the electroosmotic velocity and provides undesired adsorption sites for multiply charged molecules, such as proteins. A fused-silica capillary should be prepared for its first use by washing for 15 min each (> 20 column volumes) with 1 M NaOH and 0.1 M NaOH, followed by run buffer ( —20 mM buffer). For subsequent use at high pH, wash for 10 s with 0.1 M NaOH, followed by deionized water and then by at least 5 min with run buffer.28 If the capillary is being run with pH 2.5 phosphate buffer, wash between runs with 1 M phosphoric acid, deionized water, and run buffer.29 When changing buffers, allow at least 5 min of flow for equilibration. For the pH range 4-6, at which equilibration of the wall with buffer is very slow, the capillary needs frequent regeneration with... 

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