Abstract
With the recent developments in HPLC and the complexity of the current challenges faced by this technique the use of mobile phase gradients has become more usual. The use of gradients allows, for example, fast analysis able to separate compounds with wide ranges of hydrophobicity than possible with isocratic elution. On the other hand, the use of gradient is more demanding regarding the instrumental setup. These setups can be particularly critical in the case of short chromatographic runs in UHPLC or with the use of microbore columns on LC-ESI-MS. Depending on the type of detector and mobile phase used, very low mixing volumes can lead to excessive noise in the form of ripple in the chromatographic baseline. In contrast, a very high mixing volume can cause gradient delay and extend the time needed for column equilibration at the end of the run. Here we present a revision about some particularities regarding the use of gradient, as well as the respective caution and adjustments to minimize the chances of failure in this kind of analysis.
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