Semi-preparative and preparative (Semi-prep and prep) chromatography typically involve large injections of sample material. To properly determine the most appropriate column, you will need to consider three major points.
1. Choose the best separation mode
Choosing the best separation mode for the targeted compound is achieved by testing various analytical HPLC columns with different functional groups and from various manufacturers. Key factors to consider include,
Resolution – compound of interest must be sufficiently isolated with a good peak shape, to ensure high purity and recovery
Load – capacity of the packing material for maximum sample load
Speed – Faster is better to save time and solvent
2. Choose the prep column dimension and the particle size
Prep column inner diameter, length, and the particle size greatly affect the outcome of an efficient sample separation.
Column inner diameter – Sample load is proportional to the column cross-sectional area.
Packing material particle size – Smaller particle size can provide higher column efficiency. The draw backs are that it is generally more expensive and the back pressure is higher. Therefore, you want to choose the largest particle size possible while maintaining enough efficiency and resolution to isolate the compound of interest.
Column length – Longer column length will have better sample load and separation. However, you want to use shortest column length possible to minimize the back pressure.
3. Optimize the separation conditions
A good elution condition to try first would be the same one you use in the analytical HPLC columns. Some manufacturers provide gel materials of the same lot packed in both HPLC columns and prep columns. The exact same selectivity in analytical and prep columns allows fast and simple scale up.
When you use relatively larger particle size in the prep columns, you can increase column performance by adjusting linear flow rate and solvent composition. To further optimize the elution condition, you may want to consider trying different column length and/or particle size.
Max Load – Increase the load stepwise to determine the maximum load and the desired sample purity.
Inspect your preparative system – make sure the separation condition complies within all system parameters.