Hydrodynamic injection, electrophoresis

Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. 

Hydrodynamic injection, capillary electrophoresis, 4 633-634 Hydrodynamic lubrication regime, 15 210-211... 

Figure 9.1 Schematic representation of a basic capillary electrophoresis system. The main components include a capillary (commonly contained within a housing that allows for temperature control), a power supply, and a detector. Automation is achieved through the use of computer-controlled setting of solutions and samples, displacement forces (to replace capillary contents and for hydrodynamic injection), and automatic data collection. (Courtesy of Agilent Technologies.)...

U. Backofen, F.M. Matysik and C.E. Lunte, A chip-based electrophoresis system with electrochemical detection and hydrodynamic injection, Anal. Chem., 74 (2002) 4054-4059. 

Berzas Nevado et al. [138] developed a new capillary zone electrophoresis method for the separation of omeprazole enantiomers. Methyl-/ -cyclodextrin was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated to optimize separation and run times. Analysis time, shorter than 8 min was found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM /1-cyclodextrin and 5 mM sodium disulfide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/1 for the omeprazole enantiomers. The method was applied to pharmaceutical preparations with recoveries between 84% and 104% of the labeled contents. 

The analysis of aliphatic acids was performed using a P/ACE MDQ capillary electrophoresis instrument equipped with a 60 cm x 50 pm id fused silica capillary (Beckman Coulter, Fullerton, CA). The samples were filtered through a 0.45-gm cellulose acetate filter (Whatman, Maidstone, UK) prior to hydrodynamic injection at 15 psi for 4 s. The voltage was set to 20 kV at reversed polarity. The electrolyte, composed of 5.0 mM trimellitic acid, 50 mM tris(hydroxymethyl)-aminomethane, 1.0 mM tetradecyl-trimethylammoniumbromide, and 0.5 mM calcium chloride, had a pH of 9.8. Before use, it was filtered through a 0.2-gm cellulose nitrate filter and degassed withhelium. Detection was performedby indirect UV absorption at 220 nm. Succinic acid was used as internal standard. 

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. 

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 ... 

If the capillary is both too narrow and/or too long, it may be necessary to apply additional pressure at the injection end or suction at the detector end in order to introduce a sufficient volume in a reasonable time. For electrokinetic injection, one places the injector end of the column into the sample solution and applies a voltage across the column (for this, we will need to introduce an electrode into the sample solution). The sample ions will enter the column both by migration (at rates which will vary with their electrophoretic mobilities) and by entrainment in the EOF (at a constant rate for all ions). This difference in sample amount will cause problems in quantitative CZE work, so hydrodynamic injection is preferred in that case. In the capillary mode called capillary gel electrophoresis (CGE) (discussed in Section 13.6.4), the gel in the capillary is much too viscous to employ hydrodynamic injection, so electrokinetic injection must be used. 

Fig. 5 Electropherogram of a mixed anion standard. Hydrodynamic injection (15 cm, 120 sec, 39.1 nl) with sodium borate buffer at pH 8. Peak 1 3.2 ppm selenate peak 2 3.6 ppm selenite peak 3 1.9 ppm arenate peak 4 4.4 ppm DMA peak 5 4.7 ppm arsenite. Source From An evaluation of ultrasonic nebulizers as interfaces for capillary electrophoresis of inorganic anions and cations with inductively coupled plasma mass spectrometric detection, in Spectrochim. Acta Part with permission of Elsevier Science.

Capillary electrophoresis of PCR-amplified products is usually performed in the reverse polarity mode (negative potential at the injection end of the capillary). A coated capillary (100 mm i.d., 37-57 cm total length) is filled with a gel buffer system. PCR samples are introduced hydrodynamically or, after desalting, electrokinetically. The PCR sample and a DNA marker of known size may be injected sequentially and allowed to comigrate in the capillary. With a capillary temperature set at 20 to 30°C, separation of PCR products is accomplished at field strengths of 200 to 500 V/cm. Detection is on-line, measuring either UV absorbance at 260 nm, or LIF. 

Both injection techniques have been shown to suffer from the effects of analyte diffusion, specially when the clean capillary is initially introduced into the sample solution. Diffusion occurs across the boundary area between analyte and buffer, which is defined by the cross-sectional area of the capillary. Both techniques also suffer from the effects of inadvertent hydrodynamic flow that results from the reservoir liquid levels being at slightly different levels. While these effects are significant for the buffer-filled capillaries used in capillary zone electrophoresis, they are both much less important when the capillary is filled with a gel. 

Blittgenbach, S., Wilke, R., A capillary electrophoresis chip with hydrodynamic sample injection for measurements from a continuous sample flow. Arud. Bioanal. Chem. 2005, 383, 733-737. 

As the initially injected sample plug is normally a distance away from the capillary inlet in capDlaiy electrophoresis, the entrance region should have negligible influence on the species transport. In the region of fully developed (denoted by the subscript fd) flow field, the thermally induced pressure-driven flow causes additional hydrodynamic dispersion to the species diffusion. Analogous to Eq. 17, the effective dispersion coefficient is given by... 

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