Synchronized gradient elution in capillary liquid chromatography.

The synchronization of injection valve operation and gradient elution in capillary liquid chromatography (cHPLC) is studied. Focus is placed on the cHPLC systems which rely on the splitting of a primary flow to provide the much smaller secondary flow required at the injection device and analytical column. Owing to the tiny secondary flow rates, synchronization is necessary to achieve proper optimization of gradient elution methods. Otherwise, there is a risk of having the analytes totally or partially eluted in the initial isocratic conditions, and there is no control on the actual gradient profile reaching the column. Synchronization is first achieved by switching back the valve to bypass after injection. This is important to save time, and to avoid the gradient slope to be reduced by mixing within the internal volume of the injector (a 47% of slope reduction, in the conditions used in this work). Valve switching to bypass should be produced immediately after the arrival of the end of the sample plug to the valve (tV). Fine system synchronization is further achieved by starting the gradient at the match time (tM), which is the time required to match the arrival of both the gradient front and the end of the sample plug to the valve, and therefore also to the column inlet. Synchronization of these two events requires starting the gradient either before or even after the injection, thus to prevent a late or an early arrival of the gradient front to the injection valve, respectively. Owing to their dependence with the backpressure, both tV and tM should be measured in the presence of the column at the initial gradient conditions. Simple experiments designed to measure tV and tM are described. With synchronization according to the techniques described in this work, control on the real gradient elution conditions at the column location is maintained, the analysis time is reduced and efficiency improves. The effects of synchronization are illustrated by injecting a mixture of alkylbenzenes. At 1μL min(-1), valve switching to bypass reduced analysis time from ca. 36 to 12min (butylbenzene), and improved peak symmetry (from 2.00 to 0.94 for methylbenzene) and efficiency (the average apparent plate count increased approximately 60%). Synchronization according to the match time further improved efficiency (approximately, up to 120%).