Zone broadening

Capillary electrophoresis (CE) is sometimes called capillary zone electrophoresis because the analytes are separated into discrete zones as they migrate along the capillary. The zones are translated into peaks as the zones pass through the detector. The recorded picture of analyte peaks as a function of migration time is called an electropherogram.  

Analyte zones in CE are generally narrower than in conventional ion chromatography. Inefficient mass transfer of analytes between Uquid and solid phases, together with a curved eluent flow profile due to pumping, are major sources of zone broadening in packed columns, but these are avoided in CE. Jorgenson and Lukacs [1] showed that the theoretical plate number, N, in CE can be expressed by the following equation  

Although Eq. (11.1) is a useful guideline, additional factors will affect separation efficiency in CE. Heat generated at the high voltages employed may be dissipated more rapidly near the capillary wall than nearer to the center of the capillary. This leads to unequal migration velocities within the zone and subsequent peak deformation. For this reason, separations are often at 15-20 kV rather than at the maximum of 30 kV applied potential. 

Interaction of analyte ions with the capillary wall is another source of peak broadening. Some ions may interact with silanol groups or be adsorbed onto the wall surface. In such cases, peaks may become tailed or overly wide. These effects can be avoided or minimized by careful cleaning of the capillary and by using a more concentrated electrolyte so that electrolyte ions, rather than sample ions, will be attracted to the wall surface. 

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The smallest size difference that can be resolved is related to the pore volume, the solute shape, and the efficiency of the column (see Fig. 2.6). However, this is at very low loadings. At higher loadings the sample volume will contribute to zone broadening and may, in some cases, be the dominating factor for resolution. Thus, for fractionation, an optimum exists with respect to column efficiency (represented by the flow rate as operational parameter) and sample volume for processing a particular volume of feed per unit time. As a rule of thumb this optimum can be found at a relative sample volume of 2-5% of the column volume (Hagel et al., 1989). 

The influence of the sample volume on the zone broadening is more pronounced for media of smaller particle size (due to their inherent low zone... 

Two-dimensional separations can be represented on a flat bed, by analogy with planar chromatography, with components represented by a series of dots . In fact, zone broadening processes in the two dimensions result in elliptically shaped spots centred on each dot . Overlap of the spots is then possible, but Bertsch (30) also showed how the contributors to the overall resolution, R, along the two axes, and Ry contribute to the final resolution according to the following ... 

Figure 7.11 Separation of a vixture of PTH-anino acid derivatives by unidinensional Multiple development (right) illustrating the use of the spot reconcentration nechanisn to control zone broadening. (left). A spot size after the first development and solvent front of the second development (line). B spot area (black oval) after reconcentration by the second development. C spot size after the second development. (Reproduced with permission from ref. 117. Copyright Dr. Alfred Huethlg Publishers).

The actual temperature of separation is determined by internal and external factors. The internal factor, as it was mentioned earlier, is the generated Joule heat. The external factor is the temperature control applied by the cooling system. A temperature increase decreases the viscosity of the electrolyte and increases the diffusion of the sample, resulting in zone broadening and a decrease in efficiency. 

Moore, A.W., Jorgenson, J.W. (1993). Study of zone broadening in optically gated high-speed capillary electrophoresis. Anal. Chem. 65, 3550-3560. 

Injection variance. The injection variance is considered to be a component of the extra-column zone-broadening effects and is related to the width (u>inj) of the ideal rectangular sample plug (volume). The variance is given by... 

Other sources of variance. The brand broadening of a zone can also be caused by other sources that are not yet identified or not considered to be important at this stage of knowledge. For example, it has been shown that the coiling of a capillary in a cassette or on a spool, also has an effect on zone broadening. 

Detection in CE is performed on-column and frequently with optical detection systems. On-column detection minimizes zone broadening as... 

Similar to all chromatographic processes the band of solute that emerges from the column can be broadened by a number of processes, including contributions from the apparatus, flow of the solution through the packed bed of gel particles, and the permeation process. Corrections for this zone broadening may be made empirically it generally becomes unimportant when the sample has... 

The theoretical plate number (AO is inversely related to the amount of zone broadening occurring in a column. The greater the value for N, the more efficient is the column but differences of less than 25% are not very significant. 

Hjerten, S. (1990). Zone broadening in electrophoresis with special reference to high-performance... 

Zone broadening or electromigrational dispersion also depends on the concentration of the BGE and the ionic strength of the sample. This means that one should ideally look not only for a probe with mobility close to the analyte, but also for a BGE with a high concentration and a sample volume that is as low as possible. 

The separation efficiency in chromatography and the sample throughput in CFA and FIA are better the narrower the test substances zones. The experimental conditions must therefore be selected so that the zone broadening in the detection system is as low as possible. The zone width is conveniently expressed in terms of the time standard deviation, o, or the volume standard deviation, oy, which are related by the expression,... 

The overall zone broadening is given by the contributions from all parts of the apparatus and it holds that the variances of systems connected in series are additive... 

Hence FIA is especially suitable for rapid analyses of simple systems whereas CFA is advantageous in more complicated analyses. The theory of CFA and FIA is based on the considerations on zone broadening mentioned above (5.51) to (5.56). It still contains many serious simplifications and thus all conclusions (see [123, 148]) are semi-empirical. It has been shown [148] that, to attain sufficient... 

The width of a peak, or, in other words, the length of a solute zone, is affected primarily by diffusion phenomena leading to a broadening of the solute zone. In addition, in capillary zone electrophoresis, zone broadening can be caused by thermal effects, electrodispersion, or adsorptive effects. All these effects can be expressed as coefficients of variance cr2, adding to a total coefficient of variance of the system ofotal ... 

Under ideal practical conditions, e.g., no Joule heating and no solute-wall interactions, diffusion is the sole contribution to zone broadening. Since the flow profile in CZE is flat, radial diffusion can be neglected, and only... 

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