Gravity injection, capillary electrophoresis

Capillary electrophoresis has also been used to examine the enantiomeric purity of brinzolamide. The system consisted of a Polymicro Technologies fused silica capillary column (50 cm x 50 pm), UV detection at 255 nm, pH 2.4 buffer (28 mM heptakis-(2,6-di-o-methyl)-P-cyclodextrinand 30 mM TRIS-(hydroxylmethylaminomethane),adjusted to pH with 85% H3PO4) and an applied voltage of 20 kV. Injection of a 200 pg/mL sample was accomplished with 50 mm gravity for 10 seconds. 

Two introduction methods are commonly employed in capillary electrophoresis. Hydrodynamic injection is based on siphoning, or gravity feeding the sample into the anodic end of the capillary. The anodic end is removed from the buffer reservoir and placed in the sample solution. The capillary end is then raised so that the liquid level in the sample vial is at a height Ah above the level of the cathodic buffer, and is held in this position for a fixed time t. This process has been automated for reproducibility, and the hydrodynamic flow rate has been shown to obey Eq. 12.9 ... 

Burgi, D. S. and Chien, R.-L., Apphcation of sample stacking to gravity injection in capillary electrophoresis, Journal of Microcolumn Separations, 3,199-202,1991. 

Sample sizes in capillary electrophoresis are very small, 1-30 nl, which can be an advantage when sample volume is limited. The sample is introduced into the capillary by gravity, vacuum, pump, or electrokinetic injection. In all cases, the sample is introduced by momentarily moving the inlet of the capillary from the inlet buflfer vial to the sample vial, and then back. When gravity introduction is used, the sample is siphoned onto the column by raising the sample vial. The volume injected (F,) in nanoliters is calculated by... 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

For classical electrophoresis, samples of 0.1-1 cm are loaded into wells formed in gel slabs or layered onto the tops of gel columns, often with the addition of a sucrose solution to increase the density. For CE and CEC, much smaller samples (1-50 nl) are drawn into one end of the capillary (usually the anodic end) from a sample vial, either hydrod3mamically using gravity, positive pressure or a vacuum, or electrokinetically by applying a voltage for a short time when the EOF causes the sample components to migrate into the capillary. The reproducibility of sample injection into capillaries, typically 0.5-3%, is variable, and electrokinetic methods may discriminate between components of a mixture because of differences in electrophoretic mobilities. Time, temperature, pressure drops and sample and running buffer viscosities are all sources of variability, and automated sample injection is preferable to minimize these effects. 

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