Extra-column band broadening effects

In Sections 11.7 -11.9 we have seen how interacting parameters of column efficiency and linear flow velocity (the Van Deempter Equation), selectivity, and capacity factor (related to the distribution of analytes between phases under operating conditions) 

Little movement of the bands takes place until all the sample is on the head of the column and an ideal minimum initial bandwidth is achieved. Then the temperature is raised in GC, or the mobile phase polarity in LC is changed to more closely match the analytes, and movement and separation of these initially narrow bands begins. 

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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 chaHenging 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 extra-column band broadening effects. Pressures are lower and high strength and density PEEK plastic may be used in place of stainless steel. 

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. 

The loss in theoretical plate number by extra-column band-broadening effects should not exceed 10% (15). For a specific LC system, the maximum acceptable variance due to extra-column band-broadening can be expressed as ... 

Low-dispersion HPLC systems are necessitated by the increasing trend of using shorter and narrower HPLC columns, which are more susceptible to the deleterious effects of extra-column band-broadening. HPLC manufacturers are designing newer analytical HPLC systems with improved instrumental bandwidths compatible with 2-mm i.d. columns by using micro injectors, smaller i.d. connection tubing, and detector flow cells. A new generation of ultra-low dispersion systems dedicated for micro and nano LC is also available. 

This effect was well illustrated in a case study reported by Wu and Clausen (34). Here five alkylbenzenes were separated by an HPLC method. When this method was directly scaled to UHPLC, the separation was adequate, but the column efficiencies were lower than would be predicted. It is likely that the lost efficiency was due to the effects of extra-column band broadening. Pressure drops also did not scale as predicted. This is likely due to physical differences between the HPLC and UHPLC particles. In a similar manner to that described, it was noted that selectivity factors did not remain constant as particle size changed. For these reasons, it is emphasized that the theoretical scaling factors given earlier are approximations, and the observed relationships will likely vary from theory because of additional uncontrolled factors. Again, it is imperative that the stationary phase chemistry remain constant as the method is scaled to an alternate platform. 

It has been shown that when the intracolumn effect of mass transfer and diffusion is the main factor controlling band broadening, the column efficiency decreases with the increase of the viscosity of the meth-anol/water mixture on the other hand, when the extra-column effect is the main factor, an increase in viscosity of the eluents will help in improving column efficiency. Column efficiency is also related to the properties of the sample [86]. 

Although the above calculation is somewhat oversimplified because the effects of the compressibility of the gas have been neglected, it serves to illustrate that a reduction of the column diameter cannot be fully compensated by an increase in the column length to keep the column dispersion constant. Therefore, when narrow-bore capillary columns are to be used in GC, the extra-column contribution to band broadening will need to be reduced. 

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. 

In capillary gel electrophoresis, one of the major contributors to band broadening, besides the injection and detection extra-column effects, is the longitudinal diffusion of the solute molecules in the capillary tube [14], The theoretical plate number (N) is characteristic of column efficiency ... 

Achieving the theoretically expected performance of high-efficiency columns requires proper instrument design to ensure that band broadening outside of the column is negligible. Sources of extra-column broadening can be classified into two categories volumetric effects and electronic effects. Those associated... 

As previously indicated, this discussion is organized for chromatograms from very narrow polymer standards for which we can consider that the effect of molecular weight distribution is negligible and for which the unique separation process is size exclusion. With these limitations, the contribution to band broadening is conveniently separated into extra column effects, eddy dispersion, static dispersion, and mass transfer. In the most classical chromatographic interpretation, extra-column effects are not discussed and the three other contributions are considered as Gaussian, so there is simply the addition of their variances. The number of theoretical plates is defined as N = VJaY and the influence of v, the linear velocity of the eluent, is summarized by the so-called Van Deemter equation ... 

Section 6.2 presents various models for chromatographic columns. But it has to be kept in mind that these models only account for effects occurring within the packed bed. A HPLC-plant, however, consists of several additional equipment and fittings besides the column. Therefore, the effect of this extra column equipment has to be accounted for to obtain reasonable agreement between experimental results and process simulation. Peripheral equipment (for example pipes, injection system, pumps and detectors) causes dead times and mixing. Thus, it can contribute considerably to the band broadening measured by the detector. 

Since the model given by Eq. 6.133 concerns only the stationary phase process, the mobile phase dispersion and extra column broadening effects have to be corrected for via the deconvolution of the whole chromatogram with the peak of the imretained marker that contains the necessary information regarding these contributions to the band profiles. The resulting net chromatogram carries the contribution of the stationary phase processes only. 

Band broadening can occur in other parts of the chromatographic system as well as in the column. Contributions to this extra-column broadening may come from the injector, the detector flow cell, and the connecting tubing. Slow time constants of detectors and recorders may also contribute. These extra-column effects are more severe for early, narrow peaks in the chromatogram than for later, broader peaks. A more detailed discussion of these effects will be given in the section on instrumentation. 

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