Collection: Bare, Uncoated (Zero Flow) CE Columns

Capillary Electrophoresis

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-to-wall interactions or to modify the magnitude or direction of the electro-osmotic flow (EOF).

Bare Silica Capillaries

The capillary inner wall has a large number of surface silanol groups 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 (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 with sodium hydroxide solution (or alternatively a strong acid) followed by a 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 of 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 out 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 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 EPF. 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 derivatized 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 positively charged surface that 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 absorb in the surface is considerably 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 at low pH values. Moreover, small pH changes in the pH range 4-6 can 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 ionized 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 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).

Bare Silica Capillaries

Uncoated CE capillaries are made from high-quality drawn fused silica with a high degree of UV transparency. All Capital Analytical capillaries have an outer diameter of 375µm and are available with internal diameters of 50µm and 75µm.

For convenience of handling and easy installation, the majority of CE instruments use a cartridge, which houses the coiled capillary. In most cases, there are a number of standard lengths for each instrument type. These standard lengths are given by the way in which the capillary is coiled, i.e., each length increment should be a multiple of the coiled circumference. Capillary lengths are typically 25-75cm. Longer length capillaries i.e., 100cm or more are sometimes used for special applications such as interfacing of commercial CE instruments with a mass spectrometer.

Capillary Replacement: After the polyimide coating is removed to form the detection window, the exposed fused silica becomes extremely fragile and should be subjected to minimal handling. Many users will have experienced the frustration of breaking the capillary whilst fitting it into the cartridge unit. In order to overcome this problem, Capital Analytical can supply prepared capillaries already fitted in the cartridge aligned with the detection aperture. The exact alignment of the capillary detection window with the cartridge detection aperture is essential in order to ensure maximum UV sensitivity.

Capital Analytical offers a capillary replacement service for all popular cartridge formats such as Hewlett-Packard, Beckman, and TSP. For other instrument formats, please contact our sales office.

The injection end of the capillary should be cleanly cut to give a flat surface. Poorly cut capillaries can lead to loose polyimide coating and a cracked or shattered fused silica edge which will lead to tailing peaks, sample carry over, and a reduction in peak area precision. It is therefore of considerable benefit for the CE user to obtain expertly prepared capillaries. Capital Analytical prepared capillaries are cut to a high degree of precision with the UV detection window accurately prepared and positioned. A small length of the polyimide coating is also removed from the injection end in order to improve sample introduction precision.

Capital Analytical can also supply prepared capillaries in non-standard lengths. For customers who wish to supply their own capillaries, we offer a precision preparation service (please contact our sales office for details).