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.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC