Band broadening/spreading

Band broadening is the blurring and spreading of the boundaries of a sample zone due to diffusion. 

In a follow-up study, the same authors examined the applicability of the same device for relevant protein samples and investigated the main contributions to band broadening [82]. As a consequence of the small depth of the beds, zone spreading caused by Joule heating was shown to be negligible (see Sect. 3.1.1). Cross fields of up to 100 V/cm were applied for the separation of human serum albumin, ribonuclease A and bradykinin. The feasibility of fraction collection was demonstrated with four collected fractions of a whole rat plasma sample. Off-line analysis of these four isolated fractions by CE indicated the separation of serum albumins and globulins. 

The major source of band-broadening in CZE is longitudinal diffusion. Longitudinal diffusion refers to the axial diffusive spreading of the solute from the solute zone into the bulk solution as it travels down the capillary. The variance in peak width contributed by longitudinal diffusion is given by... 

Clearly, departures from equilibrium—along with the resultant zone spreading—will decrease as means are found to speed up equilibrium between velocity states. One measure of equilibration time is the time defined in Section 9.4 as teq, equivalent to the transfer or exchange time between fast- and slow-velocity states. Time teq must always be minimized this conclusion is seen to follow from either random-walk theory or nonequilibrium theory. These two theories simply represent alternate conceptual approaches to the same band-broadening phenomenon. Thus the plate height from Eqs. 9.12 and 9.17 may be considered to represent simultaneously both nonequilibrium processes and random-walk effects. 

Band broadening. The dilution of the sample with mobile phase during the chromatographic process. For example, a 10-ju.L volume is injected, and by the time it reaches the detector, the sample is spread throughout a 200-//.L volume. Band broadening in some cases can affect resolution of... 

A rule of thumb is that the injection volume can be as high as 30% of the volume of a peak that elutes from the column when using a small injection (e.g., 10 juL) and there should be no significant broadening of the peak with this larger injection. To understand this rule of thumb, one must consider the contributions to band broadening. At injection, the sample volume will be diluted to a volume that is mainly dependent upon the efficiency of the column. The final peak width, Wpeak, observed in the detector is a result of the volume of the sample injected and of the spreading from the column, the detector, and the extra column effects. 

When the terms contributing to band broadening are collected and equilibrium spreading is assumed to be negligible, the following general expression for overall plate height is obtained ... 

It must be stressed that the terms band or zone broadening, spreading, and dispersion used here are equivalent from the viewpoint of their physical meanings. 

Several chemical and physical variables influence the rates of band separation and band broadening. As a consequence, improved separations can often be realized by the control of variables that either increase the rate of band separation or decrease the rate of band spreading. These alternatives are illustrated in Figure 30-8. 

Band broadening The tendency of zones to spread as they pass through a chromatographic column caused by various diffusion and mass transfer processes. 

Axial dispersion, D When a band migrates along a column packed with non-porous particles, it spreads axially because of the combination effects of axial diffusion and the inhomogeneity of the pattern of flow velocity in a packed bed. This combination of effects is accounted for by a single term, proportional to the axial dispersion coefficient. It is independent of the mass transfer resistance and of the other contributions of kinetic origin to band broadening. 

Column efficiency refers to the amount of band broadening that occurs when a compound passes through the column. The typical Gaussian shape of the chromatographic band results from the random motions of the molecules making up the band as it moves down the column. The result of these random individual motions is a symmetric spread of velocities around the mean value. This spreading is referred to as molecular diffusion. 

Figure 2.4 Band broadening by longitudinal diffusion. Left Sample zone immediately after injection. It will spread out in all three axes of space (arrow directions). Right Samplezoneat a later moment. It is larger now due to diffusion and it has also been transported by the flowing mobile phase.

---