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    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.

    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.

    Learn More About Scale-Up

     


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