Capital HPLC

CE Capillaries


 CE Capillaries

The capillaries used in CE and related techniques are, almost invariably, made from drawn fused silica externally coated with polyimide for strength and mechanical stability. They normally have no coating on their internal walls. However, certain substances can be bonded to the inner surface, either to eliminate sample - wall interactions or to modify the magnitude or direction of the electro-osmotic flow.

Bare Silica Capillaries

The capillary inner wall has a large number of surface silanol roups which, when in contact with an electrolyte solution, dissociate leading to a negative charge on the capillary wall. Consequently, in order to preserve overall electrical neutrality, there must be an excess of cations in the electrolyte immediately adjacent to the inner surface (see diagram below). 
When a voltage is applied, these cations migrate in the direction of the cathode and in so doing exert a force on the surrounding fluid causing it to flow in the same direction.  This electrically induced flow is known as electro-osmotic flow (usually abbreviated EOF). In bare silica capillaries, i.e., uncoated silica, the electro-osmotic flow is high when the electrolyte pH is greater than 6 and effectively drops to zero for pH values of 3 or less.

For reproducible analyses, it is important that the surface density of free silanol groups remains constant from one analysis to the next. For this reason, the capillary is often rinsed between injections with sodium hydroxide solution (or alternatively a strong acid) followed by further rinsing with the run buffer.

For coated capillaries, the rinsing procedures are generally less harsh in order to avoid the
possibility of stripping off the inner coating of the capillary.

In order to obtain a consistent EOF, new uncoated capillaries are flushed with 0.1M
NaOH solution for 20 minutes prior to their initial use. This procedure is carried in order
to reactivate the surface silanols. The procedure must not be carried out with wall
coated capillaries.


 Electrical Double Layer - Origin of Electro-Osmotic Flow (EOF)

Capillary Regeneration

Coated Capillaries

The nature of the inner wall of the capillary can be modified by the covalent bonding of
appropriate substances. This may generate a neutral or a charged surface layer.

Neutral Coatings

Neutral capillaries are used either to reduce analyte adsorption or in situations where it is
considered desirable to eliminate EOF. Usually, the capillaries are covalently coated
with polymers such as celluloses or polyacrylamide. These types of coatings
completely eliminate EOF. Prior to the bonding procedure, the silica surface is normally
derivatised with an alkyl silane.

Charged Coatings

The capillaries can be coated either with a positive or negatively charged substance in
order to manipulate both the direction and the magnitude of the EOF and/or to reduce
analyte adsorption. For example, polyamine coated capillaries produce a highly positive
charged surface which generates, in contrast to the usual situation, a substantial EOF
towards the anode. The positive charge on the surface repels positively charged basic
compounds and their tendency to adsorb in the surface is considerable reduced.
Alternatively, the capillary can be treated to give an increased negative charge on the

surface relative to an uncoated capillary. This increase in EOF can be useful in significantly
reducing analysis times. In uncoated capillaries the EOF is strongly dependent on the buffer pH and is not present at all at low pHs. This makes it impossible, for example, to carry out micellar work a low pH values. Moreover, small pH changes in the pH range 4-6 result in relatively large
changes in electro-osmotic flow which can be a problem in terms of repeatability and precision of a method. However, if the capillary coating is fully ionised over a wide pH range, the EOF rate is then largely independent of the pH (pH independent capillary). For example, the pH independent capillary, allows the use of SDS based MECC at lower pH values than those normally used.

Low pH MECC is a particularly useful technique for the analysis of acidic solutes which at high pH are negatively charged and are therefore repelled from the negatively charged micelle.
Controlled manipulation of the surface charge density on the pH independent coating allows
a tight control of the EOF obtained for the capillary. For example, a medium charge density produces a moderate EOF rate (medium flow capillary) whilst a low level of coating generates a low EOF (low flow capillary).