Band broadening in HPLC

Farkas, T. Guiochon, G. Contribution of the radial distribution of the flow velocity to band broadening in HPLC columns. Anal. Chem. 1997,69,4592. 

The initial idea of preparing shell particles (pellicular) was to increase the column efficiency by reducing the mass transfer resistance across the particles. However, now it seems that transparticle mass transfer resistance is far from being the dominant contribution to band-broadening in HPLC (67). The columns packed with the new generation of shell particles are also enormously successful, but for other reasons (4). 

The efficiency, or plate count of a column N is often calculated as 5.54 (tr/a)2, where tr is the retention time of a standard and a is the peak width in time units at half-height.1 2 5 This approach assumes that peaks are Gaussian a number of other methods of plate calculation are in common use. Values measured for column efficiency depend on the standard used for measurement, the method of calculation, and the sources of extra-column band broadening in the test instrument. Therefore, efficiency measurements are used principally to compare the performance of a column over time or to compare the performance of different columns mounted on the same HPLC system. 

Inexplicably, phenyl glucuronide was not detected. The other peaks showed the expected retention time and elution order (13). Chromatographic efficiencies for PB-LC-MS varied from 550 theoretical plates for phenol to 8700 theoretical plates for 4-nitrophenyl sulfate. Using the HPLC-UV method, the chromatographic efficiency was somewhat better, 4300 theoretical plates for phenol and 16000 for 4-nitrophenyl sulfate. The manufacturer claims an efficiency of 7900 theoretical plates for this column. Thus, there is some added band broadening in the PB-LC-MS system. 

In HPLC peak dispersion is significantly affected by the particle size of the resin. Thus, the band broadening in a suppressor column decreases as the particle diameter of the employed resin decreases. Similarly, the void volume of the suppressor column should be as small as possible to reduce band broadening effects. An optimal suppressor column would therefore have a very low volume and would contain an exchange resin with a very small particle diameter. Both requirements are inconsistent with the analytical practice, which calls for a suppressor column that can be employed at least for one working day without regeneration. This requirement can only be met by suppressor columns with a higher volume. 

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. 

Finally, an increase in detection Knsitivity (step S) can be achieved by (1) increasing AT while holding V and constant (e.g., lower flow rates, smaller dp), (2) by decreasing k (usually by decreasing F), or (3) by reducing column diameter. With any of these approaches, the extra-column band broadening in the HPLC equipment can become important. That is, for maximum detection sensitivity, microbore -type HPLC systems may be required. 

Size-exclusion chromatography separation differs significantly fh)m other HPLC methods. For example, retention in SEC corresponds generally to values of k < 0. Similarly, there is no stationary phase apart from the stagnant mobile pha.se within the particle pores. Therefore there is no diffusion williin (he stalioniiry phase D, 0). This leads to equations for band broadening in SEC of a somewhat different form. 

Band broadening in SEC is simpler than for other HPLC methods, and as a result we can use SEC band width data to derive accurate values of the fundamental parameter p (Section V,C). For SEC, the Knox parameter B becomes [cf. Eqs. (36), (62)] ... 

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

The major advance in the way in which column eluate is deposited on the belt was the introduction of spray deposition devices to replace the original method which was simply to drop liquid onto the belt via a capillary tube connected directly to the outlet of the HPLC column. These devices, based on the gas-assisted nebulizer [5], have high deposition efficiencies, transfer of sample can approach 100% with mobile phases containing up to 90% water, and give constant sample deposition with little band broadening. 

Simple and comprehensive 2D HPLC was reported in a reversed-phase mode using monolithic silica columns for the 2nd-D separation (Tanaka et al., 2004). Every fraction from the lst-D column, 15cm long (4.6 mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles (5 pm), was subjected to the separation in the 2nd-D using one or two octadecylsilylated (Cig) monolithic silica columns (4.6 mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to lOmL/min with separation time of 30 s that provides fractionation every 15-30s for the lst-D, which is operated near the optimum flow rate of 0.4-0.8 mL/min. The 2D-HPLC systems were assembled, as shown in Fig. 7.6, so that the sample loops of the 2nd-D injectors were back flushed to minimize band broadening. 

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