Band broadening reduced parameters

Even with all these chromatographic parameters optimised, sensitivity may not be enough. The simplest way to increase sensitivity further is to inject more sample. Valve injectors are usually supplied with 20-pl loops but there are few assays for which 100-200 pi of sample cannot be injected simply by changing the loop size. A further way is to use minibore (1 mm or 2mm i.d.) or even microbore (< 1 mm) columns. Such columns give increases in sensitivity due to the narrower peaks eluted but this may reduce column loading volumes and with some equipment the extra sensitivity gained may be lost due to extra column band broadening. 

These four factors cause band broadening and work additively such that their summation contributes to H, the height equivalent to a theoretical plate. This parameter is dependent on flow rate due to the contribution from longitudinal diffusion and mass transfer. Band broadening can therefore be minimised by use of an optimum flow rate. This relationship is usually visualised in a Knox plot (Fig. 2.4) using the reduced plate height (/j) as the vertical axis and the reduced velocity (u) as the horizontal axis. The reduced velocity is given by the formula ... 

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