Making the Most of a Gradient Scouting Run

Jan 01, 2013
Volume 31, Issue 1

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?

Those of us involved in the development of liquid chromatography (LC) methods are under constant pressure from our clients to spend less time in the development process — they'd like the method yesterday! One of the challenges when we face such requirements is how to make the most of each LC run. The use of a gradient scouting run can be a very efficient way to get to the fine-tuning stage of development faster. We'll see that even if our goal is an isocratic run, starting with a gradient can be a faster way to that goal than starting with an isocratic separation. Another factor that often confuses the process today is the large choice of column and particle sizes available. Are we using a conventional 150 or 250 mm × 4.6 mm, 5-µm particle column, or an ultrahigh-pressure LC (UHPLC) system with a 50 mm × 2.1 mm, ≤2-µm column? How do we start with any of these and still get acceptable results? For the present discussion, we'll assume that a reversed-phase column will be used This is a good assumption, because somewhere around 70–80% of all methods are developed on a C8 or C18 reversed-phase column. These, and some related topics, will be covered in this month's "LC Troubleshooting."

The Isocratic Way

Isocratic 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.