Sample band broadening factors

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]. 

When sample molecules are moving down a column in the mobile phase and some are absorbed in the stationary phase, those that are absorbed fall behind those not absorbed. This causes band broadening. The factors contributing to this are related below. 

The peak recorded in a chromatogram represents the distribution of molecules in a band as it elutes from the column, the overall broadness being conveniently m sured in terms of the width of the peak. A number of independent factors such as sample-injector and detector characteristics, temperature and column retention processes, contribute to the dispersion of molecules in a band and band broadening. The cumulative effect of small variations in these factors is described in statistical terms as the variance, cr, in the elution process. Classical chromatography theory considers that the separation process takes place by a succession of equilibrium steps, the more steps in a column the greater the column efficiency with less band broadening (variance) occurring, therefore... 

Often, the limiting factor in the precision of liquid chromatographic measurement.s is the reproducibility with which samples can he introduced onto the column packing. The problem is exacerbated by band broadening, which accompanies a lengthy sample injection plug. Thus, sample volumes must be very small — a few tenths of a microliter to perhaps 500 pL, Furthermore, it is convenient to be able to introduce the sample without depressurizing the system. 

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