Pressure, electrokinetic injection

The washing of capillaries with dilute alkaline solution is advisable before analysis. The alkaline solution can be followed by deionized water and buffer. Capillaries can be washed between runs too. Samples can be introduced into the capillary by hydrodynamic and electro-kinetic methods. The hydrodynamic method applies a pressure difference (5-10 sec) between the two ends of the capillary. The pressure difference can be achieved by overpressure, vacuum or by creating a height difference between the levels of the buffer and sample reservoirs. In the case of electrokinetic injection, the injection end of the capillary is dipped into the sample for a few seconds and a voltage of some thousand volts is applied. 

The different modes of injection, i.e., gravity injection, pressure or vacuum injection, and electrokinetic injection, are addressed. Also, several practical experimental issues are described, such as correct treatment of electrolyte solutions and the importance of a rigorous capillary rinsing procedure. 

The reproducibility of the electrokinetic injection is poorer than that of the dynamic pressure injection and strongly depends on the ionic strength. Internal standards are usually added to improve the accuracy and precision [44]. For a stacking injection, a mathematical model has been developed to account for the increase in the migration time with increasing sample injection time a good agreement with the theory has been found [45]. 

Solignac, D., Gijs, M.A.M., Pressure pulse injection a powerful alternative to electrokinetic sample loading in electrophoresis microchips. Anal. Chem. 2003, 75, 1652-1657. 

The small dimensions associated with CE preclude the injection of large volumes. The sample may be introduced to the capillary either by a diplacement technique (i.e., pressure, vacuum, or siphoning) or via electrokinetic injection. The majority of commercial instruments apply a pressure differ-... 

The introduction of the samples onto the capillary column can be carried out by either displacement techniques or electrokinetic migration. Three methods of displacement or hydrostatic injection are available a) direct injection, or pressure b) gravity flow, or siphoning and c) suction. The electrokinetic injection method arose from findings that electroosmosis act like a pump (80). Both methods have advantages and disadvantages. For example, a bias has been reported in electrokinetically injected... 

Fig. 10.2. Separation of a mixture of PAHs on reversed-phase capillaries (a) without and (b) with silicate entrapment. Conditions 75 pm i.d. fused-silica capillary packed with 5 pm Nucleosil ODS particles column effective lengths 25 cm for the non-entrapped column and 17 cm for the entrapped column. Both electrochromatograms were obtained under the same conditions mobile phase, acetonitrile-0.1 M acetate buffer, pH 3.0, 80 10 (v/v) applied voltage 30 kV UV detection at 254 nm 20°C pressure 9 bar applied to both vials electrokinetic injection, 10 kV for 10s. Reproduced with permission from Chirica and Remcho [10].

Finally, the same authors reported the incorporation of electrokinetic injection into their method (31). Little interference was observed in the separation of MPA, EMPA, IMPA, and PMPA in seven different environmental matrices. Limits of detection were reduced to 1—2 xg/L for water samples, which represented an improvement of two orders of magnitude over their previous work using pressure injection. However, a sample cleanup step was necessary to reduce the conductivity of the sample to be compatible with electrokinetic injection. The samples were passed through three pretreatment cartridges arranged in series to remove sulfate, chloride, and cations, respectively. 

Two injection techniques currently in use are hydrodynamic injection and electrokinetic injection. Hydrodynamic injection is pressure driven, and therefore all components in the sample are injected simultaneously. In contrast, with electrokinetic injection, the entry of the components of the sample into the column depends on ion mobility, charge, and concentration. 

Samples are applied to the capillary by injection, but since the capillary volumes are so small (1 p.1 for a 50 pm 50 cm capillary) the sample volumes must be kept to a few nl if the resolution is not to be affected. Two modes of injections are used hydrodynamic and electrokinetic. In hydrodynamic injection, the sample vessel is connected to the upper end of the capillary, and a small quantity is introduced into the capillary either by applying positive pressure to the sample vessel, or a vacuum to the detector side. In electrokinetic injection, a low-voltage is applied for a short period while the capillary is linked to the sample vessel so that a defined quantity of sample is introduced into the capillary by electrophoretic or electroendoosmotic migration. In this case, there is a form of pre-separation, since particles with lower electrophoretic mobilities do not progress as far into the capillary as those with higher ones. 

There are two modes of injection in capillary electrophoresis hydrodynamic injection and electroki-netic injection. In hydrodynamic injection, pressure or vacuum are plaeed on the inlet sample vial or the outlet waste vial, respeetively. For electrokinetic injection, the voltage is activated for a short time with the capillary and electrode immersed in the sample. 

CC-CE Electrokinetic injection assisted with the pressure of compressed air Absorbance (19)... 

Capillary electrophoresis is conducted in capillaries filled with an electrolyte solution. Buffered electrolytes are generally used, since biomolecule mobilities and electroosmotic flow (EOF) are sensitive to pH. The ends of the filled capillaries are placed in electrolyte reservoirs that contain electrodes, and the electrodes are positioned so that electrolysis products do not enter the capillary. A small plug of solution containing the analytes to be separated is pressure- or electrokinetically-injected into one end of the capillary, and a voltage difference is applied to the electrodes such that the analytes of interest migrate toward the other end of the capillary, where they are detected. Analytes with different electrophoretic mobilities migrate at different speeds and become separated as they transverse the capillary. 

Injection volumes are in the nanoliter range to avoid system overloading, since the total volume of the capillary is in the /rl range. Direct injection techniques have been developed to ensure efficient and reproducible injection. Techniques employed are electrokinetic injection (i.e., electromigration injection), hydrodynamic injection by pressure or vacuum, and hydrostatic injection by gravity. Organic acids are almost exclusively detected with indirect UV, whereas other analytes have been measured by direct or conductivity detection. 

As mentioned before, when pressure-based sample injection is employed, the maximum injection volume must be less than the effective length of the capillary. At least 10% of the effective capillary length must remain available for separation. To inject a larger sample volume, electrokinetic injection must be employed. In most online sample preconcentration techniques, the maximum amount that can be injected without loss of separation efficiency is certainly less than the capillary volume. However, with a large volume sample injection under cathodic EOF conditions in SDS MEKC, a sample devoid of the micelle can be continuously electrokinetically injected for a volume equivalent to seven times the capillary volume without significant loss of separation efficiency under favorable... 

Several novel stable pseudostationary boundary techniques have appear, one technique dubbed moving chemical reaction boundary forms a neutral zone in the column were the H+ ions combine with the OH ions to make a water zone. This zone is stable and leads to stacking across it. A second method uses EOF balanced against back-pressure to hold the stacking boundary in a fixed location. The stability of the boundary allows for injections up to 3 h to be made. The most robust method for long injects has arguably been electrokinetic injection out of a sample vial in which the column has one Kohlrausch value and the vial a lower value. This method has shown to improve sample injection by 1000 fold. ... 

Sampling whole cells for CE involves either siphoning the cell by either applying a pressure differential or applying a potential across the capillary to electrokinetically inject the cell. In each case, a drag force is produced by the fluid flow, driving the cell into the capillary for lysis and the separation of its contents. Though these schemes are the most simplistic forms of injection, there have been several recently developed complementary techniques for introduction of cells and their contents into capillaries. 

---