Band broadening extracolumn

Experimentally there are two methods of determining the ] extracolumn band broadening of a chromatographic instrument. The linear extrapolation method, discussed above, is relatively straightforward to perform and interpret but rests on the validity.. of equation (5.1) and (5.3). The assu itlon that the individual contributions to the extracolumn variance are independent, may not be true in practice, and it may be necessary to couple some of the individual contributions to obtain the most accurate values for the extracolumn variance [20]. It is assumed in equation (5.3) ... 

The band broadening and the analyte dilution resulting from extracolumn band broadening were compared between the microfluidic chip system and the conventional macro-scale system. For a proper comparison, we calculated the analyte concentration at the peak maximum of the bioactive peaks (Cmax) from E-64 injections in both systems. It turned out that the dilution factor when comparing the concentration at peak maximum with the injected concentration was only 10% higher for the microfluidic chip system in comparison to the conventional macro-scale system. 

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 is the property of the column. Ideally, all analytes separated on the same column should demonstrate the same number of theoretical plates. Secondary equilibria of the analyte in the column or extracolumn band broadening can alter this. 

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... 

Detectors used in HPLC should have low internal volumes to minimize extracolumn band broadening in addition, they should be sensitive and should respond quickly to concentration changes. Few detectors fulfill all of these requirements. One of the oldest detectors used in HPLC is the refractive index detector, which detects subtle differences between the refractive index of the pure mobile phase and a mobile phase containing the solute. This detector is universal, i.e., it can detect any solute whose refractive index differs from that of the pure solvent. However, its sensitivity is poor, which practically precludes its use in trace analysis. Besides, refractive index detectors are very sensitive to changes in the composition of the mobile phase and to temperature flucmations. The former makes their use in gradient elution impractical the latter requires that the detector is thermostated to at least 0.01°C. 

There are two general experimental methods for estimating the extracolumn band broadening of a chromatographic instrament. The linear extrapolation method is relatively straightforward to perform and interpret but rests on the validity of Eq. (1.34) and the model used to calculate the contribution for the column variance. A plot of a T against tR, Vr or (1 + k) for a series of homologous compounds will be linear. The true column efficiency can be obtained from the slope of the line and o ext from the intercept on the vertical axis [162,167,168]. The assumption that the individual... 

Careful consideration must be given to the design of the detector flow cell as it forms an integral part of both the chromatographic and optical systems. A compromise between the need to miniaturize the cell volume to reduce extracolumn band broadening... 

The main disadvantages of packed-column suppressors are that they require periodic regeneration due to limited capacity result in significant extracolumn band broadening. 

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. 

---