"Capillary for CE is typically manufactured to tolerances of +/- 2 ^m.

"Capillary for CE is typically manufactured to tolerances of +/- 2 ^m.

mon in commercially available capillary, selecting different pieces of capillary nominally 20 ^m in diameter could give peak areas that vary substantially. Cutting pieces from a large spool of capillary is no guarantee of uniformity because the diameter may vary along the length of the spool.

The injection plug length in Table 1 is the linear distance within the capillary that is occupied by the injected sample volume. An injection plug that is too long may result in wide bands and loss of resolution, particularly if the analytes cannot focus. Increasing the capillary diameter allows the injection of substantially larger volumes of sample without increasing the plug length. Because the volume of a cylinder increases with the square of the radius a doubling of the capillary diameter will allow the injection of four times as much sample without changing the plug length.

The viscosity of the fluid being delivered is the most difficult parameter to know with accuracy. The aforementioned examples have assumed the viscosity of water at 25°C. Within the range of temperatures typically used in CE separations (15-60°), the viscosity of water varies in a nonlinear manner from 1.138 to 0.467 centipoise. Almost anything added to the water will alter both the viscosity and the temperature-viscosity relationship. The addi tions of macromolecules, such as cellulose derivatives or acrylamide polymers, are extreme cases. These molecules display remarkable viscosity behavior with changes in flow rates. Linear polymers, for example, can extend and align themselves when forced through a capillary. They can show a reduction in viscosity with increased flow. Other systems may increase in viscosity with flow because of polymer entanglement. Even systems as simple as methanol-water can show complex behavior. For example, a 1:1 mix of methanol and water has a higher viscosity than either pure solvent. It is also important to remember that when calculating volumes injected into fluid-filled capillaries, the viscosity of the fluid in the capillary is usually more significant than the viscosity of the sample (unless one is analyzing highly viscous samples).

Entering the Poiseuille equation into a spreadsheet program simplifies fluid delivery calculations such as these. There is also a Windows-compatible computer program called "CE Expert" that can perform these calculations (this program is currently available at no cost directly from Beckman Coulter, Inc.).

1.3.2. Sample Handling

Four basic strategies are used to deliver these fluid volumes into capillaries: Positive pressure, vacuum, gravity, and electrophoresis. Positive pressure and vacuum have been the most common methods of filling and rinsing capillaries. Positive pressure up to 100 psi (7 bar) has been used for rinsing. This pressure is delivered either from a source of compressed gas, such as nitrogen, or from an on-board air pump that applies pressure to the headspace of a buffer reservoir. Vacuum delivery is limited to 10 psi or less but can be useful for drawing fluid from containers that cannot be made pressure tight.

Gravity and electrophoresis are not practical for filling and rinsing capillaries, but they are used, along with pressure and vacuum, for injecting samples into capillaries. Gravity injection (sometimes called hydrostatic injection) is accomplished by inserting the inlet end of the capillary into the sample vial and raising the vial and capillary relative to the outlet end. Reproducible gravity injection requires that the vial be raised to the same height for the same duration of time at each injection (gravity itself being a fairly reliable source of motive power). Pressure and vacuum injections are more complicated. Regardless of how the pressure differential is created, a finite time is required for the pressure to reach a steady state. Changes in pressure on the order of 0.05 psi can be significant at the low pressures commonly employed (0.1-1 psi). Systems that rely on pressure or vacuum

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must have some sort of feedback mechanism to compensate for these variations. These systems either adjust the delivered pressure or the delivery time to maintain the desired product of pressure and time. In well-engineered systems a 3-s injection at 1 psi and a 10-s injection at 0.3 psi should give identical results, because both are 3 psi-second injections. In practice the longer, lower pressure injection usually gives better performance because it allows a longer time for the system to respond to variances.

Electrophoretic or electrokinetic injections do not conform to the Poiseuille equation. In this method of sample introduction, the inlet of the capillary is inserted into the sample and the outlet into a buffer vial. Voltage is briefly applied. Through a combination of electrophoresis and electroosmotic flow sample is drawn into the capillary. This technique is valuable when delivering sample to a gel-filled capillary or when pressure delivery is not possible. There is a possibility of bias when using this technique (29). Components that migrate more rapidly in the electrical field will be over-represented in the sample compared to slower moving components. To maximize the volume injected, the sample should be at a considerably lower ionic strength than the run buffer. Subsequent injections will show reduced peak areas because each injection delivers salts from the capillary buffer into the sample vial, raising the ionic strength of the sample. This effect can be minimized (and injected quantity increased) by pre-injecting from pure water immediately prior to the sample injection.

Most commercial systems employ some sort of carousel or X-Y-Z robotic system to move the buffer and sample vials to the capillary. These systems may also provide refrigerated storage for labile samples. In this case the sample storage temperature should be regulated independently of the capillary thermostatting system.

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