The use of a gradient scouting run can be a powerful tool to jump-start method development. But what conditions should be chosen, and what do we do with the results?
The Isocratic WayIsocratic methods, where the mobile-phase concentration is constant throughout the run, are the technique of choice for most chromatographers. This is because such methods tend to be more intuitive to develop and adjust, they don't require waiting for the column to equilibrate between runs, and artifact peaks from the mobile phase are less of a problem than in gradient elution. The age-old technique to develop isocratic methods is quite simple. Just start at a high percentage of the B, or organic, solvent (usually acetonitrile or methanol), step back in 10% increments until you obtain a promising chromatogram, and then fine-tune it. So, 90% B, 80%, 70%, and so forth. This tried-and-true technique has been in use as long as modern LC has, but even under the best circumstances, it may take half a day or more before you have a glimpse of potential isocratic conditions, and you may be thoroughly disappointed when the polarity range of your samples is so large that a single isocratic run may not be possible.