Band-broadening

There are many reasons for band broadening and it is important that these are understood and the phenomenon kept to a minimum so that the number of theoretical plates in the column is high. 

The mobile phase passes in a laminar flow between the stationary phase particles (Fig. 2.3). The flow is faster in the channel centre than it is near a particle. The arrows in Fig. 2.3 represent mobile phase velocity vectors (the longer the arrow, the greater the local flow velocity), Eddy diffusion and flow distribution may be reduced by packing the column with evenly sized particles. 

The first principle on which a good column is based is that the packing should be composed of particles with as narrow a size distribution as possible. The ratio between the largest and the smallest particle diameters should not exceed 2, 1.5 being even better (example smallest particle size 5 pm, largest particle size 7.5 pm). 

The broadening due to eddy diffusion and flow distribution is little affected, if at all, by the mobile phase flow velocity. 

Third Cause Sample Molecule Diffusion in the Mobile Phase 

The second principle is that the mobile phase Uow velocity should be selected so 

Eddy diffusion is caused by variable size chaimels through which the solute molecules pass. In wider channels the molecules travel faster than in narrow channels and therefore give rise to band broadening the shape of the soUd phase particles determines the actual effect of eddy diffusion on band broadening and can be held to a minimum by using spherical particles of uniform size and packing them evenly into the column. The degree of eddy diffusion is similar at all flow rates. 

Longitudinal diffusion refers to diffusion of solute molecules in the direction of flow and, since solute diffusivity is low in Uquids (providing that the analysis time is not excessively long), it is a neghgible problem. 

Mass transfer problems arise from limitations in the rate of diffusion of solute molecules between the stationary and mobile phases. In particular, molecules that diffuse more deeply into the stationary phase will lag behind those which diffuse less deeply. This will be accentuated by high flow rates and can be best minimised by coating the stationary phase thinly over a non-porous particle. However, such pellicular packings suffer from the disadvantage that there is a relatively large, chromatographically inactive core which reduces the loading capacity of the column. 

Extra-column band broadening is caused by either excessive dead space within the chromatographic system (e.g. large internal volume fittings, tubing, detector cells) or alternatively by an inefficient method of sample introduction. It is therefore important to use low, or better 

zero dead volume fittings and to have the injection device as near as possible to the column. 

If one component migrates through the column in the mobile phase only, with no interactions with the stationary phase, the migration time is called Im- An analyte with interactions with the stationary phase will be retained and will elute at Ir  

The can be determined by injecting a component known to have no interactions with the stationary phase. 

Time units can also be replaced with volume units  

If the distribution of each band is assumed to be a Gaussian distribution, the extent of band broadening can be expressed by the column efficiency N  

Another expression for the band broadening in a column with length L is the plate height H  

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Assuming a Gaussian profile, the extent of band broadening is measured by the variance or standard deviation of a chromatographic peak. The height of a theoretical plate is defined as the variance per unit length of the column... 

Schematics illustrating the contributions to band broadening due to (a) multiple paths, (b) longitudinal diffusion, and (c) mass transfer.

One contribution to band broadening in which solutes diffuse from areas of high concentration to areas of low concentration. 

To determine how the height of a theoretical plate can be decreased, it is necessary to understand the experimental factors contributing to the broadening of a solute s chromatographic band. Several theoretical treatments of band broadening have been proposed. We will consider one approach in which the height of a theoretical plate is determined by four contributions multiple paths, longitudinal diffusion, mass transfer in the stationary phase, and mass transfer in the mobile phase. 

One contribution to band broadening due to the time required for a solute to move from the mobile phase or the stationary phase to the interface between the two phases. 

The electroosmotic flow profile is very different from that for a phase moving under forced pressure. Figure 12.40 compares the flow profile for electroosmosis with that for hydrodynamic pressure. The uniform, flat profile for electroosmosis helps to minimize band broadening in capillary electrophoresis, thus improving separation efficiency. 

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

Band broadening is also affected by the gradient steepness. This effect is expressed in Table 16-14 by a band compression factor C, which is a fnuctiou of the gradient steepness and of equilibrium parameters. Since C < 1, gradient elution yields peaks that are sharper than those that would be obtained in isocratic elution at

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The results from the overload of the more polar solute are similar to that for the aromatic hydrocarbons, but the effect of the overloaded peak on the other two appears to be somewhat less. It is seen that there is little change in the retention of anisole and acetophenone, although the band width of acetophenone shows a slight increase. The band width of benzyl acetate shows the expected band broadening... 

In order to achieve the best efficiency the SEC column should be operated at optimized operating parameters. The most important ones are flow rate [cf. van Deemter equation for band-broadening effects (21)], sample viscosity (depends on molar mass and concentration of the sample), and injection volume (7). 

Sample loading must be reduced in accordance with the column inside diameter. Polymers exhibit high solution viscosity, and in order to avoid band broadening due to viscous streaming the sample concentration must be reduced for narrow-bore columns. Overloading effects become noticeable at much lower concentrations using 4.6-mm columns compared to 7.5-mm columns because of the effective sample concentration in a smaller volume column. 

The contribution of the equipment between injection unit and detector cell should be negligable in relation to the column for a sufficient column characterization short connections with narrow capillaries and zero dead volume unions are the precondition for reliable plate numbers. Every end fitting of a column causes additional band broadening. In the past a column type was offered that could be directly combined without any capillary links unfortunately, it has disappeared from the market. 

This equation is based on experience with liquid chromatography of low molecular weight samples displaying single peaks. Its application for the GPC of polymers, however, contains a disadvantage, as it mixes two inseparable properties the retention difference for the separation and the peak width for the contrary effect of band broadening. Such a procedure is acceptable if both effects are accessible for an experimental examination. For the GPC experiment, we do not possess polymer standards, consisting of molecules that are truly monodisperse. Therefore, we cannot determine the real peak width necessary for a reliable and reproducible peak resolution R,. This equation then is not qualified for a sufficient characterization of a GPC column. 

Because the polydispersity of the polymer is reflected in the width of the chromatographic peak, we require that the column band broadening contribution to the peak width be minor compared to that from the polymer itself. This criterion cannot always be met. 

For acrylic polymers produced via emulsion polymerization, a set of two or more 30-cm-long columns with 10-ju,m or less packing material will usually ensure that the observed polydispersities are minimally influenced by column band broadening. 

Traditionally, column efficiency or plate counts in column chromatography were used to quantify how well a column was performing. This does not tell the entire story for GPC, however, because the ability of a column set to separate peaks is dependent on the molecular weight of the molecules one is trying to separate. We, therefore, chose both column efficiency and a parameter that we simply refer to as D a, where Di is the slope of the relationship between the log of the molecular weight of the narrow molecular weight polystyrene standards and the elution volume, and tris simply the band-broadening parameter (4), i.e., the square root of the peak variance. 

Figure 2.5 Schematic representation of a loop-interface scheme for concunent eluent evaporation. The sample is first loaded in a loop and then, after switching the valve, directed by the caiiier into the GC column. The solvent evaporates from the front end of the liquid, thus causing band broadening. Since the column is not flooded, very large amount of liquid can be inti oduced.

The non-intrusive manipulation of carrier gas effluent between two columns clearly has significant advantages in two-dimensional GC. In addition, a pressure-driven switch between the columns introduces no extra band broadening to an eluting peak. 

D. Tong and K. D. Battle, Band broadening during mobile phase change in unified cliromtography (GC-SEC) , 7. Microcolumn Sep. 5 237-243 (1993). 

The flow profiles of electrodriven and pressure driven separations are illustrated in Figure 9.2. Electroosmotic flow, since it originates near the capillary walls, is characterized by a flat flow profile. A laminar profile is observed in pressure-driven systems. In pressure-driven flow systems, the highest velocities are reached in the center of the flow channels, while the lowest velocities are attained near the column walls. Since a zone of analyte-distributing events across the flow conduit has different velocities across a laminar profile, band broadening results as the analyte zone is transferred through the conduit. The flat electroosmotic flow profile created in electrodriven separations is a principal advantage of capillary electrophoretic techniques and results in extremely efficient separations. 

Figure 9.2 Pressure-driven (a) and electrodriven (b) flow profiles. Laminar flow in pressure-driven systems results in a bullet-shaped profile, wliile the profile of electroosmotic flow is plug-shaped, wliich reduces band broadening.

Separation in column 1 (C-1) removes early-eluting interference compounds, and so considerably increases the selectivity. The fraction of interest separated in C-1 is then transferred to column 2 (C-2) where the analytes of the fraction are separated. These transfers can be carried out either in forward mode or backflush mode. The forward mode is preferred because the backflush mode has two disadvantages for polar to moderately polar analytes. For most polar compounds, it leads to additional band broadening, while for more retained analytes there is a decrease in the separation obtained earlier in the process (31). 

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