Scaling up reversed-phase flash chromatography methods is often necessary as reaction scale increases. This is especially true when other non-chromatographic purification techniques do not work or meet purity and/or yield needs.
While preparative reversed-phase chromatography is an option, this technique suffers from low loading capacity and slow throughput. This post details how to simply scale-up reversed-phase flash purification methods using automated flash chromatography systems and prepacked columns.
For success scaling reversed-phase flash chromatography methods, several operating parameters must be held constant…
These parameters are determined during small-scale method development, typically using columns 6 to 60 grams in size. Once a method is optimized on the small column, scale-up is linear based on the ratio of column media masses. For example, if 140 mg of a mixture is successfully purified on a 12-gram C18 column then a 60-gram column will successfully purify 5x that amount, or 700 mg, Figure 1.
Figure 1. Linear scale-up purification of a 7-component mixture. Top - 12 g Biotage Sfar C18 column with a 140 mg load. Bottom - 60 g Biotage Sfar C18 column with a 700 mg load (5x more than the 12 g column).
For this example, the seven previously mentioned operating parameters were held constant, Table 1.
| Parameter | Biotage® Sfär 12 gram | Biotage Sfär 60 gram |
| Sample concentration | 350 mg/mL | 350 mg/mL |
| Sample load % | 1.17% | 1.17% |
| Column brand/media | Biotage Sfär C18 | Biotage Sfär C18 |
| Elution solvents |
MeOH/water |
MeOH/water |
| Elution gradient |
30% MeOH (1 CV) 30-100% MeOH (10 CV) 100% MeOH (2 CV) |
30% MeOH (1 CV) 30-100% MeOH (10 CV) 100% MeOH (2 CV) |
| Linear velocity (cm/min) | 9.2 | 9.5 |
| Detection | DAD UV 190-400 nm | DAD UV 190-400 nm |
Table 1. Chromatographic operating parameters
By maintaining these seven parameters, purification scale-up is possible even to kilogram scale.
For more information on scale-up please download our whitepaper Optimizing Chromatographic Purification for Rapid Results in Chemical Manufacturing.