Reducing Cycle Time Using Gradients

In 2001, Valko et al. reduced the column length to only 50 mm and increased the flow rate to 2mLmin [42]. The gradient time was diminished to 2.5 min with a gradient cycle time of 5 min. Measurement of CHI and evaluation of log P were excellent with a 3-fold improved productivity. In these conditions, the system dwell volume (Vd) becomes essential and only dedicated chromatographic devices with Vjy lower than 0.8 mL can be used [42]. Special attention should be paid to the injected volume, which must remain lower than 3 pL to avoid any overloading or extra-column volume contributions. 

The use of high flow and fast gradient HPLC has gained a lot of popularity because of the ability to reduce LC/MS/MS cycle times during bioanalysis. In the case of fast gradient HPLC, peak shapes were improved and method development times were minimized, especially when multiple analytes with diverse functionalities had to be separated. Flows as high as 1.5 to 2 mL/min were achieved on a 2.1 x 30 mm Xterra C18 column.7 Details are discussed in a recent review.8... 

Several conclusions were drawn from these experiments. First, the decrease in analyte signal experienced at higher flow rates was consistent with analyte band dilution in accordance with the known concentration dependence of ESI. Second, these data confirm that high-flow gradient elution can be used to significantly reduce LC cycle times without an unduly sacrifice of chromatographic fidelity. It is noted, however, that LC cycle times did not decrease proportionally with flow rate and that eventual deterioration occurred at... 

The speed and throughput of the fast HPLC approach were increased by use of shorter columns with a reduced internal diameter, Zorbax SB Cig (3.5 ixm, 30 X 2.1 mm i.d.) (Hewlett Packard, Waldbronn, Germany). These columns were used on the same instrumental hardware with the same mobile phase compositions at a flow rate of 2.0 mL/min with a temperature of 40°C. However, the linear gradient profile was completed in 2 min and held at this composition for 0.1 min, yielding an overall cycle time of 2.6 min. 

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