Extracolumn effects

How is the efficiency influenced by the BGE Peak I broadening is the result of different processes in CZE I occurring during migration [in addition, extracolumn effects contribute to peak width (e.g., that stemming from the width and shape of the injection zone, or the -f. aperture of the detector cell)]. If the system behaves I linearly, the individual peak variances (the second mo-9 ments), o j, are additive according to a... 

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... 

Quality of column (efficiency) and extracolumn effects such as dead volumes also contribute to peak broadening. For all these reasons, it is very difficult to make an accurate prediction of Wh unless data from the same column and compounds are used as a starting point. 

What we would like to know is the influence of the extracolumn effects relative to those inside the column. Let us first calculate the peak width and the variance of a peak inside the column ... 

Figare 3.8 Extracolumn effects apparent plate count obtained on columns with different internal diameters as a function of the retention factor. The extracolumn band spreading was assumed to be 15 iL. A significant deterioration of the performance is observed for the smaller-diameter columns. 

Both the mobile-phase flow and the sample should be distributed evenly over the entire cross section of the packed b. This should be accomplished in a minimal volume to minimize extracolumn effects. How important a good distribution of the sample is depends on both the column performance and its aspect ratio. It is more of a problem with short and wide columns than with long and thin columns. This is why a good sample distribution is more of a concern in preparative columns than in analytical columns. However, measurable effects are observed for high-performance columns if the diameter length ratio exceeds 0.05. An example would be a 4.6-mm x SO-mm column packed with i-paa particles. 

Extraeobum Effects Extracolumn effects are a more significant source of peak-shape problems than most people think. As a matter of fact, there is barely a chromatogram in the world that is free from extracolumn effects, but we have come to accept them as part of the reality of chromatography. Only when these effects become excessive do we attempt to reduce them. Also, extracolumn effects may cause problems when method are transferred from one instrument to another or from a standard column to a microbore column. 

The previous section dealt with extracolumn effects originating in the fluid path of the HPLC instrument. A different type of extracolumn effect is associated with the electronics of the instrument the time constant of the detector, the sampling rate of the A/D converter, and the digital filtering. All of these factors may contribute to a distortion or broadening of the peak in a similar way as do volumetric effects. The only difference is that they operate in the time domain rather than in the volume domain therefore it is best to express their influence in time units. 

The primary purpose of a column testing procedure in the context of column packing is to verify the quality of the packed bed. This information can be used to optimize a packing procedure or to verify the quality and reproducibility of an already established packing procedure, llierefore we need a procedure that is sensitive to differences in the packed bed, but insensitive to otW parameters such as extracolumn effects or the chemistry of the packing. the test procedure should provide results fairly quickly. We can therefore write down a few requirements for such a testing procedure ... 

The peak volume should be large enough to be free from distortions by extracolumn effects. This is best accomplished by an adjustment of the retention factor of the test compound. 

Figure 173 Influence of extracolumn effects on a chromatogram. The early-eluting peaks exhibit increased peak tailing and low plate counts. (The chromatogram obtained without extracolumn effects is shown in the background.)...

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. 

Although ideally the observed variance is equal to the column variance, most HPLC systems detract from the column efficiency. Equation (22) can be used to calculate the importance of extracolumn effects ... 

Injection effects as a result of mass and volume overloading or the injection technique can detract from column efficiency. As with other extracolumn effects, injection effects become more critical with smaller bore columns, which require smaller injection volumes and low flow rates refer to Reference 110 for a discussion of extracolumn effects in microcolumn systems. 

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