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…
- Sample concentration
- Sample load as a percentage of stationary phase mass
- Column media and brand
- Elution solvents
- Elution gradient in terms of column volumes
- Solvent flow velocity (linear velocity, not flow rate)
- Detection wavelengths
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.
Table 1. Chromatographic operating parameters
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% MeOH (1 CV)
30-100% MeOH (10 CV) 30-100% MeOH (10 CV)
100% MeOH (2 CV) 100% MeOH (2 CV)
Linear velocity (cm/min) 9.2 9.5
Detection DAD UV 190-400 nm DAD UV 190-400 nm
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.