The Instrumental Gradient Delay Dwell Time

The dwell time is best measured under realistic conditions, for example, with %B = 0-100% in 15 min. Instead of the column a capillary with preferably small internal diameter is used, in order to build up a realistic back pressure while increasing the volume as little as possible. Eluent A is water, eluent B water with a few parts per thousand of acetone. When the UV absorbance is set to zero, eluent B should have a maximum absorption of slightly less than 1 AU. The gradient is 

During tj, the analytes move with different speeds and with an individual retention factor k at the initial conditions %B through the column  

Substances with long retention times are relatively unaffected, on the other hand substances with short retention times and small retention factors A g are strongly affected. 

The mobile time is the sum of an isocratic fraction tQ and z gradient fraction to g. (Note The mobile or dead time remains of course overall unchanged, because whether 100% water or 100% acetonitrile is used is irrelevant for the elution of an true inert component, which is influenced only by the column dimensions and the flow. In reality, there is a slight influence  

With decreasing kg the substances have moved further into the column. Therefore, tfl g is different for each substance and only the remaining part of the column is available for the gradient. Of course, the predetermined instrumental gradient slope does not change, but the effective gradient slope does, as the column available for the gradient has become shorter.  

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