Stationary phases gradient method

In situ loading during the chromatographic development. By this method a continuous gradient of the hquid stationary phase is created on the PLC layer in the direction of the mobile phase flow. This is achieved by developing the plate with a suitable multieomponent solvent system. 

Modem technologies provide many techniques for expanding the throughput of an analytical laboratory. The task that needs to be accomplished and the possible drawbacks should be carefully considered. Optimized LC equipment can utilize columns packed with much smaller stationary phase particles to achieve significant reductions in gradient time while still achieving the same or even better peak capacities than conventional methods. 

Their method included a Waters 2795 Alliance HT (high throughput) HPLC system with an integrated autosampler. The stationary phase was a Supelco C18 column (250 x 4.6 mm, 5 fim). The mobile phase consisted of solvent A (water containing 2mM ammonium acetate and 0.1% formic acid) and solvent B (methanol containing 2mM ammonium acetate and 0.1% formic acid). The mobile phase was delivered at a flow rate of 0.6 mL/min in a step gradient mode 50% solvent B from 0 to 0.4 min and 100% solvent B from 0.4 to 0.8 min. 

Epirubicin — Anthracyclines have been used in cancer chemotherapy for more than 30 years and epirubicin (EPI) is one of the most widely used agents.55 Li et al. developed a high-throughput method for the analysis of epirubicin in human plasma using UPLC-MS/MS.56 A Waters Acquity UPLC system was coupled with a Micromass Quattro Premier MS. The stationary phase was a Waters Acquity BEH C18 column (50 x 2.1 mm, 1.7 jum). The column was maintained at 30°C. Solvent A was 0.1% formic acid in water and solvent B was acetonitrile. The mobile phase was delivered at a flow of 0.20 mL/min in a step gradient mode at 85% solvent A at 0 min, 70% solvent A at 1.00 min, and 85% solvent A from 2.50 to 4.00 min. Epidaunorubicin (EPR) was the IS. 

It is estimated that over 65% (possibly up to 90%) of all HPLC separations are carried out in the reversed-phase mode. The reasons for this include the simplicity, versatility, and scope of the reversed-phase method [23]. The hydrocarbon-like stationary phases equilibrate rapidly with changes in mobile-phase composition and are therefore eminently suitable for use with gradient elution. 

The approach to method development is similar to the one described for HPLC and can be characterized as a rapid stationary phase screen using column and solvent switching with gradient elution followed by development of an isocratic preparative method. SFC has been successfully applied to the analytical and preparative separation of achiral and chiral compounds. 

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