Band broadening gradient elution

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 most common mobile phase is a gradient of petroleum ether or hexane with increasing concentrations of acetone or diethyl ether. Development of the column should be optimized for each sample to afford a quick and effective separation to avoid band broadening. The separation can be followed visually. The most non-polar a- and 3-carotenes are eluted first as a yellow band followed by the chlorophylls and other more polar carotenoids like cryptoxanthin, lutein, and zeaxanthin that frequently fuse together and appear as a single band. ... 

In cases where mass transfer is rapid, as is the case with most small molecule separations, then isocratic elution can offer advantages such as automatic fraction reprocessing and solvent recycle. However, with larger synthetic objectives the rate of mass transfer is comparatively low so isocratic elution leads to band broadening and subsequently to recovery of the peptide at high dilution. Most preparative HPLC based peptide separations are carried out under gradient and overload conditions that allow for maximum throughput in terms of time and quantity. 

The behavior of chromatographic columns operated in gradient elution, under linear conditions i.e., assuming linear isotherms for all the solutes) has been studied theoretically by numerous authors [2,4-10]. The most comprehensive treatment is that based on the linear solvent strength (LSS) theory of Snyder et al. [5,6]. This theory has formd widespread acceptance [7,8] and has been extended to include the contributions of the various mass transfer resistances to band broadening [9-11]. It assumes the injection of infinitesimal pulses of a feed and a linear gradient of the volume fraction of a mobile phase modifier, cf). 

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. 

Band broadening during a gradient-elution run arises in the same way as for isocratic separation, with only minor differences (24,39). Band wid A is... 

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