Bob Bickler

How do I decide between normal- or reversed-phase flash column chromatography?

December 30, 2019 at 4:49 PM / by Bob Bickler

Choosing a good purification strategy is an important for successful crude compound purification. A major factor in your strategy is choosing between normal-phase or reversed-phase chromatography.  How do you choose?

In this post, I will provide some simple guidance on helping determine which route to take.

In my role at Biotage, I am frequently supplied with compound structures from chemists and then asked – how do I purify this? So, with that question I open a dialog with the chemist to understand purification goals and learn more about the compound’s properties.

Where I like to start is with the question of solubility – in which solvents is your compound most soluble.  This typically tells me whether normal- or reversed-phase flash chromatography is most likely to work.

A rule of thumb I use is if the sample is organic solvent soluble (DCM, EtOAc, toluene, ether, etc.) then try normal-phase.  If the crude material is soluble in polar solvents (alcohols, DMSO, DMF, acetonitrile, etc.) then I first suggest reversed-phase.  If the sample is hydrocarbon soluble (hexane, heptane, cyclohexane) then I think reversed-phase, but normal-phase may also work. Table 1 provides my suggested purification routes based on crude sample solubilty.


Solvent Normal-phase Reversed-phase
Water   X
Methanol   X
Acetonitrile   X
Ethanol   X
Propanol X X
Acetone X X
Ethyl acetate X X
Ether X X
Toluene X X
Hexane (and other saturated hydrocarbons) X X

Table 1. Sample solubility and flash purification route

The reason I lean towards reversed-phase for the lipophilic compounds is that they usually have little functionality other than C and H, which can make them difficult to retain and separate on silica. Compounds that are organic soluble are also candidates for reversed-phase chromatography but should be dry loaded.

Organic amines provide a different challenge and, as many of you know, these compounds, especially if basic, are quite difficult to purity.  With organic soluble 2° and 3°organic amines and N-heterocycles, I steer away from silica to amine-functionalized silica as the chromatography is usually simpler and better.  For primary amines and quaternary amines, these often are soluble in polar solvents so I suggest reversed-phase for their purification.

As with most anything, there are always exceptions to the rule, but more times than not following these solubility guidelines will provide you with the right purification route.

How do you determine which purification route to use? If you're looking to learn more about flash purification, be sure to read my last white paper on "How To Be Successful in Flash Chromatography":

Read The White Paper

Topics: Normal phase, Reversed-phase

Bob Bickler

Written by Bob Bickler