Band broadening extracolumn effects

E. Extracolumn Band Broadening or Variance To maximize the effective number of theoretical plates, the contribution of the entire chromatographic system to band broadening (system variance, o-2ys) must be minimized. The system variance may be broken down into contributions from the column variance, a 01, as described above, and extracolumn diffusion and mixing processes, cr2x. As with the case of the column variance, extracolumn variance is an additive property and may be broken down into the major components ... 

Efficiency, N The column efficiency characterizes the combined effects of the sources of band broadening due to axial dispersion and mass transfer resistance. It is derived from the width of the elution peak observed as the response to the injection of a small, narrow pulse of a dilute solution of a compoimd. It is difficult to correct for the contribution of the extracolumn sources of band broadening which have to be kept small. In preparative and nonlinear chromatography, there is a correlation between the colmnn efficiency and both the steepness of the shock layer and the duration of the band beyond the retention time However, the column efficiency is essentially a concept of linear chromatography, and it is difficult to extend to and use in nonlinear chromatography, except through the shock layer thickness concept. 

Today, Fast LC is the easiest way to increase HPLC laboratory productivity by 3-5 fold without sacrificing resolution, sensitivity, or assay reliability.19 Figures 3.18-3.20 demonstrate the versatility and dramatic speed achievable with Fast LC. Chapter 4 discusses the effect of extracolumn band-broadening... 

If the sum of these various contributions to band broadening produces a band of width w,-, when the column is disconnected from the separation system, an additional term must be added to Eq. (5-2) L/16(R/iv,). Extracolumn effects are most important in high efficiency separations where sample retention volumes are small, e.g., in narrow diameter columns. The initial sample bandwidth (a) can be controlled by minimizing... 

Considering a chromatographic process controlled by a partition equilibrium and neglecting extracolumn effects (i.e., band broadening caused by factors outside the column, e.g., tubings, detector etc.), several factors can contribute to the overall solute band broadening eddy diffusion, longitudinal diffusion, and resistance to mass transfer in mobile and stationary phase. 

It is clear from Equation (20) that peak volumes are directly proportional to the volume of the column and that samples elute with smaller peak volumes from the same column when filled with a more efficient, that is, smaller size, packing material. The more efficient the column, the narrower are the sample bands and the more important is the effect of extracolumn band broadening. Wider columns provide for more peak volume, and this reduces the importance of extracolumn band broadening. 

HPLC detectors Like GC, HPLC has a wide variety of detectors, universal or specific, destructive or nondestructive, mass flow or concentration responsive, and with even more challenging requirements for interfacing to spectrometers in hyphenated techniques. Tubing of even smaller bore than described earlier in item 5 is necessary to connect the effluent end of the column to the detector to avoid extracolumn band-broadening effects. Pressures are lower, and high strength and density PEEK plastic may be used in place of stainless steel. 

Conventional GC instruments are inadequate. When short capillary columns are used at high flowrates, the effects of extracolumn sources of band broadening are amplified, and injection plug width, detection method, and data-processing techniques may contribute to large reductions in system resolving power. 

FIGURE 5.5 Golay plots for a 5.0-m-long, 0.25-mm-i.d. thin-film column using hydrogen carrier gas showing the effects of extracolumn band broadening defined by the total instrumental dead time Af. A binary diffusion coefficient of 0.4 cm /s and a retention factor of 2.0 are assumed. 

These trends toward the use of shorter columns with narrower diameter mean that the overall volume of today s UHPLC columns can be much smaller than the HPLC columns of the past. Consequently, the extra-column band-broadening effect on efficiency can become especially significant in UHPLC as the effect is closely related to column dimensions (14,15). Recently, extra-column band-broadening has been extensively investigated in UHPLC (16-19), conventional HPLC (20), capillary HPLC (21), and modified convenfional HPLC (22). Additionally, the effects of extracolumn volume on other chromatographic parameters including retention factor, selectivity, and pressure drop also become more significant in UHPLC. 

As shown in Figure 3.8, an increased injection volume can have a negative effect on the column efficiency. Therefore, the injection volume should be appropriately scaled down when an HPLC method is transferred to a UHPLC method or vice versa to achieve the same sensitivity and avoid overloading or detector saturation and extracolumn band-broadening. The scaling factor is mainly based on column dimension, as shown in Eq. (3.26). However, lower injection volumes than calculated can often be used on UHPLC to achieve the same sensitivity due to enhanced peak heights from use of the high-resolution columns and low carryover from the injector ... 

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