How do I choose between Normal- or Reversed-phase flash column chromatography for my compound purification?

How to choose between normal- and reversed-phase flash column chromatography is an excellent question and one that my readers often ask.  Those who use column chromatography know that as long as the reaction products or compounds are fairly non-polar and near neutral pH they will have successful purifications.  However, when your mixture's chemical characteristics are more challenging (polar, non-polar, basic, acidic) there are other options that are available to successfully separate pure compounds.

In this post, I will discuss the criteria you can use to guide your choice between normal- or reversed-phase flash chromatography.

There is nothing more important to purification success with chromatography as compound or sample solubility. Concentrating on just the most popular solvent system of hexane and ethyl acetate may send you down the wrong purification path (is nothing ever easy with chromatography?).  Well, in this case it can be easy with just a little chemistry knowledge. If you need to purify a synthetic reaction mix, then you already know your product’s structure as well as those of your starting materials. So, the main question is - in which solvent did you perform the synthesis? The answer to this question can guide your flash purification decision.

Organic Synthesis

How, you may ask. Think about it.  If your synthesis is performed in DCM, CHCl3, or other relatively non-polar solvent, then your compounds are likely lipophilic but with some polar functionality and amenable to normal-phase chromatography.  If your reaction solvent is DMSO or DMF, the products created may be lipophilic but getting them out of the polar solvent, may be a challenge so why not simplify the purification by using reversed-phase?  If the reaction products are polar, definitely use reversed-phase. As always, try normal-phase TLC with various solvent systems to verify your the proper purification mode.  If your compound of interest has a normal-phase TLC Rf under 0.5, then it is likely amenable to silica flash column chromatography.  Above 0.5, I would look at reversed-phase.

Natural Products

A good rule of thumb is if the sample or compound is soluble in DCM, EtOAc, or similarly water-immiscible solvents then normal-phase is likely the best approach. If the compounds are soluble in water-miscible solvents then reversed-phase is likely the best way forward. Alkane soluble compounds – what do you think? (answer later in the post)

In the natural product world, base your flash purification choice on extraction solvent choice – DCM, EtOAc, and other non-water miscible solvent extracts will most likely contain compounds best purified by normal-phase. Alcohol and water extracts are best purified by reversed-phase. Hexane extracts will likely contain the most lipophilic compounds which, in many cases, will not retain and separate well on silica so I typically use reversed-phase, sometimes even non-aqueous reversed-phase to separate these compounds, e.g. carotenoids.

In the case where non-polar or even moderately polar solvents (e.g. acetone) are used as extraction solvents, again, let TLC be your guide.  If any of the extracted compounds run with the solvent front or have Rf values > 0.5 in 100% hexane (or heptane, cyclohexane, etc.) then consider reversed-phase.  If you achieve separation of compounds in 50% or less EtOAc (in hexane, heptane, or other similarly lipophilic co-solvent) then pursue a normal-phase purification solution.  Above 50% polar modifier, I suggest reversed-phase.  Remember if it doesn’t stick to silica it will stick to C18 (a commonly utilized reversed-phase sorbent). Likewise, if it sticks to silica too well, reversed-phase will likely do a better job of separating the compound from its impurities. As a reference I have summarized these suggestions in the table below.

Normal-Phase - Reversed-Phase Selection Criteria

What approach do you take to chromatographic mode selection?

Is there anything you have learned about choosing either reversed- or normal-phase that can help educate other readers?

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