Chemists are often taught how to use glass columns in schools, so they take fundamental background experience into industry which provides an intuitive advantage in process development applications. However, with the exception analytical RP-HPLC, there is usually relatively little experience of preparative scale reversed phase purification.
Reversed phase flash chromatography is a very effective purification technique. Its main application areas include polar, ionizable and highly lipophilic compounds which cannot easily be separated by normal-phase techniques.
Unlike normal phase chromatography, reversed phase uses a hydrophobic stationary phase (e.g. C18 or ODS) and hydrophilic mobile phases (methanol/water, acetonitrile/water). By converting the silica’s active, polar silanols sites to neutral, lipophilic sites, compounds that will either aggressively stick to silica or not stick at all can be retained, separated and eluted using water-based solvent systems. Thus KP-C18-HS, or spherical 30 micron PR HP-Sphere C18 silica is more like RP-HPLC than traditional glass column flash chromatography ever was.
For that reason, it is sometimes difficult to visualize the best conditions to run or scale-up reversed phase systems, especially when transferring purification methods, where normal phase methods are typically used, and where gut feel intuition counts for a lot.
Remove the Guesswork....
We show how to use flash columns to scale-up reversed phase chromatography. Three different scale up situations are modelled and we’ve included useful experimental parameters for each one as a template for various scale up situations.
Experiments in our study were conducted with sample loadings ranging from 80 mg to 2.7 g using three different flash cartridge sizes. They were first equilibrated with 4 CV @20% Acetonitrile. The solvent gradient used was 1 CV @ 20% Acetonitrile, then 10 CV @ 20–70% Acetonitrile, and finally 1 CV @70% Acetonitrile (i.e. 30% water).
Summary
In this example 80 mg of a 4 component test mix was separated using a 12 g Biotage® SNAP KP-C18-HS cartridge. After equilibration of the column (using 4 CV of 20% acetonitrile in water), a standard reversed phase gradient was run using acetonitrile/ water. The same gradient and conditions were applied to larger cartridges on scale up (1 CV 20% Acetonitrile, 10 CV 20–70% Acetonitrile, 1 CV 70% Acetonitrile), using larger loads and with similar results.
A typical starting point for reversed phase flash chromatography is a binary gradient, going from a low to high fraction of organic, typically with water as co-solvent. Before the C18 chains can be used effectively it is important to equilibrate the column, transitioning out of the dry state or organic storage solvents, and into a higher percentage of aqueous solvents to start the run.
We demonstrated a 33-fold scale increase in both column and load during the reversed phase purification run, which enabled the separation of 80 mg–2.7 g. Typically reversed phase flash columns are loaded to 0.5–1.0% wt, but this is dependent upon sample composition. For easily resolved compounds, it is possible to increase loading to more than 1% by weight of silica.