How to prevent compound precipitation during flash column chromatography

Compounds precipitating during flash chromatography is at best an inconvenience when working up your crude reaction mixture.  Precipitation during purification typically happens in the column or in the tubing exiting the cartridge.

In this post, I will propose a strategy that can minimize and perhaps prevent this issue from occurring.

In my 40 year career as a chromatographer I have both encountered this issue personally and have heard myriad organic synthesis chemists complain of this problem, perhaps you too have had to deal with this.

This problem occurs when one or more compounds in the crude sample have different solubility when being purified by chromatography than they do when they are part of the reaction mixture.  During purification, the compounds separate from each other and must remain in solution as the solvent blend becomes increasingly polar (if using normal-phase flash chromatography). Just because your reactants and reaction products may be soluble in the reaction solvent does not mean they all will be soluble in the mobile phase, especially as they become more pure during chromatography.

As mentioned in a previous post, understanding reaction mixture solubility is the first step in optimizing your purification so it makes sense to add a drop of your crude in various solvents to see what “crystalizes” (I love puns! Sorry about that). Once you have determined crude solubility and performed TLC, your method is then transferrable to your flash system.  However, the possibility of precipitation still exists as “pure” product may remain soluble during the solubility test (influenced by other compounds in the crude) but when separated are no longer are soluble.

There are two options to eliminate this potential problem

1.       Dry loading, which essentially precipitates all of the crude components on a sorbent allowing them to be selectively solvated and eluted during purification

2.       Use a mobile phase modifier.  This option I discussed previously in regards to pH adjustment in reversed-phase chromatography, but it can be implemented for normal-phase as well.

Implementing a modifier can be a simple strategy to ensure compound solubility.  You can either add a co-solvent to your individual mobile phase solvents or, if using a Biotage® Isolera system, make use of its additive feature, Figure 1.  With a third and fourth solvent reservoir available, the Isolera provides the ability to pump a third solvent isocratically (constant %) during the gradient while allowing your primary solvent reservoirs to remain unaltered.

Figure 1. Including a co-solvent additive into a gradient can help prevent compound precipitation during purification. Isolera Spektra systems provide this capability.

Incorporating a co-solvent can help prevent untimely crystallization during the run and save you the headache of trying to recover your valuable compound from a packed cartridge or tubing.

How have your dealt with the precipitation issue?

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Published by

Bob Bickler

Technical Specialist, Biotage

4 thoughts on “How to prevent compound precipitation during flash column chromatography”

  1. During my PhD days, I was dealing with urea compounds that had terrible solubility. They refused to dissolve in anything else than DMF or DMSO. When running flash columns they were streaking massively (nitro compounds, so you could already see the mess). This was quite surprising to me as on TLC I was getting nice round spots. I remember I read somewhere, that when you choose to dry load your sample you’re circumventing solubility issues and your sample doesn’t have to even be soluble in a mobile phase. So I did alittle experiment and injected a mobile phase saturated with my mixture. You can imagine that was very little compound and the run was rather analytical than preparative. I got perfect looking chromatogram. So obviously solubility plays a major role. If your mixture components are poorly soluble in the mobile phase you’re very limited with your prep. No solubility, no chromatography.

    1. In the old days of flash chromatography powered by rubber bellows, my reaction mixtures, dissolved in CH2Cl2, often crashed out soon after they hit the mobile phase containing hexane. Dry loading is a godsend for this. A 1g sticky brown lump, dissolved in CH2Cl2 then dry loaded onto 4g of silica has an enormous surface area compared the original lump. This massive increase in surface area means the entire sample comes into intimate contact with the mobile phase and is far easier to dissolve. If you expect problems try CH2Cl2 instead of hexane, you will need a lot less EtOAc though.
      Of course some compounds are brick dust and not suited to prep chromatography, just analytical. On rare occasions I’ve dry loaded and the front of the peak comes off as expected but tails for most of the gradient! It’s not “eluting”, it’s “dissolving” off the column. There’s no shame in recrystalising.

      1. Hi Derek,

        You too are correct in suggesting dry loading as a means to get around compound precipitation during flash chromatography. The situation you describe is also the scenario for including an additive, which can be used when dry loading as well to help speed up the solvation and minimize the tailing.

    2. Hi Tomasz,

      You make a good point – dry loading is an excellent means of inhibiting compound precipitation during the run as all of the compounds are in a dry form on a porous sorbent and will only dissolve when the correct mobile phase mixture is attained in the gradient.

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