![]() These components would include, but may not be limited to, injector needle seat, injection valve, capillary tubing, heat exchanger(s), detector flow cell, etc: If such data is made available, one can refer to manufacturer specifications to find the internal volumes of the HPLC components that contribute to extra-column dispersion and add the individual contributions together. We know of at least three different methods to measure extra-column volume, which we describe here: This is impossible (at least with current technology!) so it is important to assess the extra-column volume and minimise it whenever possible. ![]() Where σ column 2 is peak dispersion due to mechanisms inside the column, and σ EC 2 is peak dispersion due to extra-column volume effects.Īn ideal chromatographic system would only exhibit minimal dispersion inside the column, and extra-column diffusion should be non-existent. This mathematical description of the peak is convenient because it makes it possible to describe peak width (more precisely, retention time variance) as a sum of contributing factors: It is common to describe a chromatographic peak as an idealised Gaussian function, with retention time being the maximum of the curve, and peak width being measured by standard deviation. Collectively, the volume of these components is known as extra-column volume, V EC. Diffusion occurs in the internal volume of the injection system, as well as the detector, the connecting tubing, and any inline filter, guard column, or other devices present in the flow path. As described by Van Deemter in 1956, mechanisms inside the column include eddy, axial, and longitudinal diffusion of the solute molecules as they flow through the packing material, as well as the mass transfer of solutes into and out of the stationary phase.īroadening outside the column is due to molecular diffusion, which happens from the point the molecules are injected until they are detected. Several factors contribute to the broadening of a chromatographic band, occurring both inside and outside of the separation column.
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