BIOMARK002.239 - Scale-up reversed phase header

Bob Bickler

How do I scale-up my reversed-phase flash chromatography method?

April 27, 2020 at 2:53 PM / by Bob Bickler

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.

12g and 60g comparison

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.

Learn More About Scale-Up

Topics: Chromatography Fundamentals, Reversed-phase, Scale-Up

Bob Bickler

Written by Bob Bickler