Slow injector-to-column sample transport to maximize resolution in liquid chromatography: Theory versus practice.

A simple chromatographic model including extra-column sample bandspreading is built and validated experimentally. It is used to quantify the advantage of slowly transporting under isocratic elution of the injected sample band along the pre-column volume of the chromatographic system compared to the conventional injection method at constant flow rate. For fast analyses (<30 s), the model predicts a maximum relative gain of 20% for the isocratic peak capacity of weakly retained compounds (k < 1) at the price of a 60% increase in retention time (+20 s) for short (3.0 cm long) narrow-bore (2.1 mm i.d.) columns packed with sub-2 μm particles and for low-dispersion (∼2 μL extra-column volume variance) vHPLC systems. These predictions were confirmed experimentally using a 2.1 mm × 30 mm column packed with 1.9 μm XBridge-C for the rapid separation of small molecules in 25 s at 40 °C using a mixture of methanol and water (75/25, v/v) as the eluent. For longer analyses (>30 s), the model allows for the determination of the optimum and slow injection speed that maximizes the trade-off between the analysis time and the isocratic peak capacity. For 2.1 mm × 20 mm columns packed with 2 μm particles and a highest retention factor of 10, the optimum injection speed is about 10-15% of the operating column flow rate for a maximum relative gain of 5% in overall isocratic peak capacity at constant elution time. Overall, the proposed injection method is directly applicable for enhanced resolution of weakly retained polar compounds in RPLC without the need of changing separation conditions, which may strongly increase the retention times of the late apolar eluters.