Fast Gradient Elution in RPLC

The initial configuration proposed by Valko et al. for log P gradient determination was based on a gradient cycle time of about 15 min with a 150-mm column [40]. This procedure was modified by Mutton, who stated that resolution could be maintained when the gradient time and/or column length were reduced or the flow rate increased [59, 60]. 

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. 

Donovan and Pescatore described another fast-gradient approach with very short columns (20x4.6 mm internal diameter) packed with a porous polymer (known as ODP columns) [38]. This chromatographic support presents a high chemical stability and can be used at pH 2, 10 or 13 to analyze neutral analytes. This procedure allowed a relatively high flow rate (2mLmin ) and a gradient from 10 to 100% methanol in only 7 min. The mathematical treatment was simplified and based on the direct transformation of retention time to log P. For this purpose, two standards (toluene and triphenylene) were used to minimize retention time variations from run-to-run and instrument-to-instrument, and to facilitate the 

Nevertheless, this method was successfully applied by Gulyaeva et al. for the log P and log D determination of 15 P-sympatholytic drugs [56]. Another study by Welerowicz and Buszewski compared the HpophiHcity values of P-blockers obtained with a column made of a monoHthic-silica Cjg with a conventional porous silica particles Cjg as reference material [27]. A modified method was used for evaluating logP with two main differences (i) logfeg was considered rather than retention times, and (ii) benzene and butyl-benzene were used as calibration compounds. 

In this study, chromatographic experiments were 10 times faster with the monolithic column and results were equivalent to those obtained with the silica-based columns. This approach could be further optimized with faster gradient since flow rate should be increased by a factor 3 or 7 compared to conventional Cig supports [61, 62] and gradient time reduced by the same factor [63] to fully exploit the potential of monolithic supports. 

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