We are often asked by customers performing a new analysis in Flash: what is the most efficient starting point to develop an isocratic or gradient method?
There is no universal answer, but a good starting point prior to beginning flash chromatography is to ensure your compounds have an Rf value of at least 0.2 to 0.3 with TLC analysis.
| Starting Percentage B | Ending Percentage B |
|---|---|
| 2% | 100% |
| 5% | 95% |
| 5% | 75% |
| 10% | 50% |
If you do, a scouting gradient is an excellent starting point to begin to create and optimize a method. Below are some typical linear scouting gradients that can be used where we reference only the strong solvent (B). Generally, a shallower gradient will provide the greatest resolution, but the law of diminishing returns also applies here. Simply put, you don’t want to broaden your peaks too much, or have too much separation between them, this separation between peak elution is otherwise known as a retention factor or k. You should target a K* of 2-8 for complex samples.*
When determining the run time, this can vary based on how many compounds you are trying to separate, as well as the degree of difficulty. However, 10-15 Column Volumes (CV) at 1CV per minute, will be more than enough for most separations. Based on your screening results, if all compounds elute at ≤ 25% of the gradient then the analysis can be achieved isocratically. If the compounds elute at ≥40% then a gradient system is best.
In between 25% to 40%, it is up to the operator to determine if an isocratic or gradient profile is best.
Starting Solvent Suggestions for TLC and Flash Chromatography
- Polar compounds: 100% EtOAc or 5% MeOH/dichloromethane
- Normal compounds: 10-50% EtOAc/Hexane
- Nonpolar compounds: 5% EtOAc/Hexane, 5% ether/Hexane, 100% hexane
Reverse Phase Flash Chromatography
- Acetonitrile
- Methanol
- THF
*K prime definition the period that the sample component resides in a stationary phase relative to the time it resides in the mobile phase. It is calculated from the retention time divided by the time for an unretained peak