John W. Dolan

The first step in getting an acceptable separation is to get the retention times in the right general region (3). This often is accomplished by changing the mobile phase organic content (%B) for a reversed-phase separation in 10% increments from 100% organic until the retention factors k are in the range of 1–20, or better yet, 2–10. Because peaks move in a regular fashion as the mobile phase strength is changed, we can generalize the effect of solvent strength as The Rule of Three. This states that the k-values change by approximately threefold for each 10% change in organic. Because k changes three times per 10% change, a 20% change will result in approximately 3 × threefold, or roughly 10-fold. Using such guidelines and a little common sense, usually we can find isocratic conditions that give us the 1 < k < 20 or 2 < k < 10 retention times in three to five runs. Wouldn't it be nice to be able to find these conditions in the very first run? Well, with the help of a gradient scouting run, you usually can. The use of a gradient scouting run to speed isocratic method development is the subject of this month's "LC Troubleshooting."