The bane of organic synthesis for most chemists is purification rather than synthesis. Synthetic reaction mixtures are rarely devoid of impurities so some type of purification is necessary. Most often flash chromatography is used but for many chemists, it is less well understood than their chemical reaction and provides some level of anxiety.
In this post, I will summarize the five most important steps to creating a successful flash chromatography method and thus the anxiety associated with it.
The seminal paper on adsorption flash chromatography by W. Clark Still in 1978 (Still, Kahn, & Mitra, 1978) explained how flash chromatography, when used following their published guidelines, provides fast (10-15 min) reaction mixture purification for compounds moderately well separated (ΔRf ≥ 0.15). In the 40 years since Still’s paper was published, development of newer synthetic organic chemistry methods has led to reactions that often create complex mixtures. These reaction mixtures can be challenging to purify, requiring different purification methodology such as gradient elution, and potentially even alternative media to isolate a pure target product.
In my 40 years of practicing chromatography (GC, ion, HPLC, TLC, flash) I have learned there is an art, as well as science, involved with chromatography. The art is a bit subjective but the science is real and tangible. While there are no really universal chromatography methods, there are 5 specific science-based steps chemists should follow, and understand, to increase their success.
- Understand your chemistry. Basically, this means to know your mixture’s solvent compatibility/solubility and the target molecule’s possible reactivity toward silica or other media. You do not want your target to react with another solvent or the chromatography media (e.g. organic amines with silica)
- Your solvent choices will guide you to the next decision – whether to use normal- or reversed-phase purification as detailed below…
- Neutral/acidic reaction mixtures soluble in low to medium polarity organic solvents are likely candidates for silica columns and normal-phase chromatography
- Polar solvent (alcohols, acetone, acetonitrile, etc.) soluble mixtures and those reaction products that are ionic/ionizable likely require reversed-phase flash chromatography
- Organic soluble secondary, tertiary, and heterocyclic amines are best separated and purified using amine-functionalized media
- Spend some time developing a suitable purification method. With normal-phase flash purification, including those requiring amine-functionalized media, thin-layer chromatography (TLC) is almost always a good place to start. TLC enables you to evaluate different solvents and solvent mixtures to find those providing the best separation of your target molecule from the reaction by-products. For reversed-phase methods, method development can be performed using either HPLC or a reversed-phase flash column (they are re-usable)
- Optimize your method further by considering a step gradient. Step gradients can improve column loading capacity (sample load/throughput) while reducing solvent consumption and purification time. Two TLC runs using the same solvents but at different ratios will generate scalable purification methods
- Linear method scaling. Though most synthetic chemists are not concerned with purification scale-up, those in scale-up or kilo-scale facilities will be and may use your method as a starting point. Successful low-scale (mg or g) purification methods are scalable based on column size ratios but not on flow rate. For this, linear velocity must be equal (or close). Still suggested a linear velocity of ~5 cm/min. You may find this a bit slow, especially if using smaller particle media. The key with scale up is to maintain the same elution method and loading % (expressed as grams of purified material over grams of media) while adjusting flow rate to maintain linear velocity on larger flash columns.
For information that is more detailed you may want to listen to my webinar on this subject.
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