Impact of instrument and column parameters on high-throughput liquid chromatography performance

The speed and separation performance of high-throughput liquid chromatography (HT LC) was investigated. We evaluated the contributions of various experimental parameters to the total analysis cycle time, including column length (column void time), gradient delay, flow rate, auto-sampler (A/S) speed, and software related delays. The best case injection-to-injection cycle time of 22 s was achieved using 12 s gradient time and 2.1 × 20 mm columns packed with 1.7 μm C18 particles. The total 22 s analytical duty cycle consisted of 2.5 s column void time, 1.8 s gradient delay, 12 s gradient time, and approximately 5.7 s for software setup delay time that served as column re-equilibration time. The achieved peak capacity for an alkylphenone sample was approximately 35, giving a peak production rate of 95 peaks per minute. We estimated the impact of LC system dispersion on peak capacity and peak production rate (peak capacity per unit of time). For HT LC scenarios (5-50 mm columns and 4.8-120 s long gradients) we observed that even a minor amount of system dispersion (2 μL2) reduces the achievable peak capacity and peak productivity rate significantly. HT LC-MS analysis using 2.1 × 5 mm guard column with duty cycle of 22 s was successfully demonstrated.