Electrokinetic injection, method

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... 

Traditionally, electrokinetic injection is the most common method for sample injection and separation into the microchannel utilizing some form of electroosmotic pumping. However, depending on the sample, due to the different mobility in an electric field, a bias to anionic, neutral, or cationic analytes is possible. Careful adjustment of pH and ionic strength can reduce this effect [2]. A broad injection band and sample leakage phenomena are also important defects of the electrokinetic injection method. Both are known to reduce the separation efficiency of a device since they result in a wide sample distribution within the microchannel and an increasing signal baseline as the number of injection runs increases [3-6], respectively. Other injection methods, like pressure injection, do not show this method-dependent effect. 

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 injection volumes in CE are extremely small because of the use of capillaries with very small diameters. Typical injection volumes are in the order of 10—50nE (a fog droplet is +10 nL). Injection of such small volumes of sample into the capillary is very challenging and requires specific approaches including use of rotary-, split- and micro-injectors, electrokinetic and hydrodynamic injection. Although all these injection techniques have shown to be quite appropriate, electrokinetic and hydrodynamic injection methods are mostly applied. Recent commercial instruments are usually equipped with these two injection modes as standard methods.Chapter 3 provides more details on the different injection modes. 

Hydrodynamic injection was compared with electrokinetic injection (data not shown). The two injection modes gave comparable percent peak areas. Electrokinetic injection gave slightly higher resolution compared to hydrodynamic injection. For the CE-SDS method, electrokinetic injection is generally recommended. 

Sample injection in NCE is very important for reproducible results with low limits of detection. In spite of some development in NCE very little effort has been made to develop sample injection devices in this technique. Of course sample injection in NCE is a challenging job due to small volume requirement [87], The controlled injection of small amounts of sample is a prerequisite for successful analysis in NCE. Electrokinetic injection (based on electroosmotic flow) is the preferred method and Jacobson et al. [88] optimized sample injection using this approach. Pinched injection allowing injection in minute quantities [89,90] and double-T shaped fluidic channels [91] have also been used for this purpose. Furthermore, Jacobson et al. [92] used a single high voltage source to simplify instrumentation. Similarly Zhang and Manz [93] developed a narrow sample channel injector to improve... 

In most cases, sample introduction on-chip is achieved using electrokinetic (EK) flow [3]. Two important EK injection modes, namely, pinched injection and gated injection, have been developed. Furthermore, some alternative injection methods are described. 

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. 

The separation capillary is prepared by polymerizing 6%T, 5%C monomers with 30% formamide and 7 M urea inside a 37 cm long, 50 pm i.d. fused silica capillary. Electrokinetic injection at 200 V/cm for 30 s was used, since hydrodynamic injection does not work with gel-filled capillaries. Separation on the gel occurs by seiv-ing, so that the shortest fragments elute first. Figure 12.12 shows the data obtained using a TAMRA dye label that is excited at 543.5 nm and emits at 590 nm. This method has a detection limit of 2 zmol (1 zmol = 10 21 mol) for each fragment. 

In previous work of the same group (72), the electrokinetic injection of DNA fragments was optimized as well by means of a simplex method. CGE-LIF was also used. In this case, BGE concentration, sample injection voltage, and time were the factors to be optimized. The optimum conditions were reached after only nine experiments. Figure 6.5 shows the spatial evolution of the simplex method used in this work (the initial tetrahedron (vertices 1-4) and the subsequent movements of reflection and contraction). Vertex 9 was considered as the optimum for injection of the Ikbp DNA ladder (l.OmM TTE buffer, 20s injection, 55V/cm electric field injection). 

Die injection system must be capable of reproducibly introducing very small sample volumes into the capillary. The volume of the whole capillary is only in the order of p,L. To minimise band broadening, sample plugs must be as short as possible. Hence, not more than a few nL of sample are introduced into the capillary. Two injection methods are commonly used (1) electrokinetic injection and (2) hydrodynamic injection. 

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. ... 

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