Fast gradient separation by very high pressure liquid chromatography: Reproducibility of analytical data and influence of delay between successive runs

•The performance of five methods used to implement fast gradient separations are compared.•Reproducibility of peak area, retention time, peak width is presented.•Conditions include constant flow rate, two inlet pressures, and column-wall temperature.•An original programmed flow nearly constant pressure method is included.•The influence of the length of the post-run times on the reproducibility is also discussed.

Five methods were used to implement fast gradient separations: constant flow rate, constant column-wall temperature, constant inlet pressure at moderate and high pressures (controlled by a pressure controller), and programmed flow constant pressure. For programmed flow constant pressure, the flow rates and gradient compositions are controlled using input into the method instead of the pressure controller. Minor fluctuations in the inlet pressure do not affect the mobile phase flow rate in programmed flow. The reproducibilities of the retention times, the response factors, and the eluted band width of six successive separations of the same sample (9 components) were measured with different equilibration times between 0 and 15 min. The influence of the length of the equilibration time on these reproducibilities is discussed. The results show that the average column temperature may increase from one separation to the next and that this contributes to fluctuation of the results.