Not Voodoo Home / Chromatography Notes / Comments?
Problem: My compound is not stable to silica gel (if you want to find out if your compound is stable to silica, see Troubleshooting: TLC). How can I purify it by chromatography if it decomposes on silica?
Solution: If the separation is an easy one, you could try purifying on florisil (200 mesh) or alumina. For more challenging separations, it is possible to deactivate silica gel (reduce its acidity) so that it is less damaging to your compound. To deactivate silica gel, prepare a solvent system containing 1-3% triethylamine. You will use less of the polar component of your solvent system than usual. Pack your column using this system and flush with solvent equaling the volume of the silica. Discard the eluant. The silica should be deactivated, and you can run the column with either the triethylamine system or with your usual solvent system.
Problem: Your compound should be off the column by now, but you're still collecting fractions with no sign of it.
Solution: A number of things could have happened here.
Problem: I cannot separate the components of my reaction mixture using column chromatography, even though there is a large Rf difference. All the fractions are mixed. I don't understand why this is happening.
Explanations and Solutions: One reason for this behavior is that you are being misled by thin layer chromatography. You see two compounds, but one may be the degradation product of the other, and the degradation occurs on silica gel. During a column, this process goes on during elution, and all fractions have both product and degradation product. Check that your compound is stable to silica.
Sometimes the wrong choice of solvent will also encourage this behavior- if the compound with low Rf dissolved well in the eluant, and the compound with high Rf does not, you may observe this behavior. Try to find a solvent system that dissolves both compounds well.
Problem: My compound is very nonpolar and does not have an Rf as low as 0.3-0.4 in any solvent system. What do I do?
Solution: If it's an easy separation, a high Rf might not be a problem. If it is, you will want to consider other purification methods. If the compound is a solid, you may be able to purify by crystallization, or you are working on large scale, you could use distillation. If none of these methods work, but your compound is mostly pure, think about the future of the compound. Are you about to convert it to a more polar species that will be easier to purify? Depending on the chemistry, sometimes it's possible to go on to the next reaction with your mostly-pure compound and purify successfully at a later stage.
Problem: My compound does not have any major impurities in it and I hate chromatography. Do I have to run a column on it?
Solution: It depends on the next step of the reaction sequence. The best answer is to try the next step without purification, on a small scale, to see how it goes. Another option is to run the compound through a short plug of silica, which will remove baseline impurities and call allow you to skip the fraction-collecting stage of chromatography.
Problem: My compound is very polar: it does not run up the plate even eluting with 100% EtOAc. How do I purify it by chromatography?
Solution: You could use reverse-phase silica for your column (link), or try more aggressive solvent systems to move it off the baseline. Solvent systems containing ammonia can be useful in this context: prepare a stock solution of 10% ammonium hydroxide in methanol, and try using 1-10% of this in dichloromethane for very polar compounds. Do not exceed 10% of the stock- that much methanol might dissolve your silica.
Problem: I have less than 50 mg of compound, how do I purify it?
Solution: You can run a miniature flash column by packing a short (6') pipette with cotton, sand and silica, just like you'd run a normal column. Choose a solvent system that places your compound at an Rf of ~0.2. You will have to add solvent to the column every 1-2 fractions, but it's simple to apply pressure with a pipette bulb, and you'll be finished in no time. This method works better than you might expect.
Problem: Your compound takes FOREVER to come off the column, it starts coming off at a reasonable point in the elution, but it doesn't stop coming off until many many fractions later.
Solution: When the compound starts coming off the column, increase the polarity of your eluting solvent. If there are no impurities at lower Rf, you can increase it significantly and avoid the annoying trailing effect. It is important to keep the same solvent system, and just increase the percentage of the polar component.
Problem: I have two compounds very close in Rf, how do I separate them?
Solution: Although it's not very much fun, the best way is to use solvent gradient during elution. You begin with a solvent system in which your compound runs at an Rf of about 0.2 (or less) and increase the percentage of the polar component of the solvent system slightly each time you replenish the solvent in the column. You will need to experiment with degrees of gradient for each difficult case before you find conditions that give optimal separation. Some people find it's easier to run two columns instead of one. For difficult separations, you will often have some mixed fractions- rather than throw them out or contaminate pure fractions, keeping mixed fractions "for a rainy day" is recommended. You can always purify your mixed sample later, if need be.
Problem: Your crude reaction mixture is not soluble in the solvent system you plan to use to elute your column. You don't know how to load it on the column gracefully. (note: this happens most often with the ethyl acetate/ hexane solvent system, and usually is only a problem on large reaction scale.)
Problem: Your compound has clogged the silica somehow- elution is very slow
Explanations and "Solutions": This is not very common, but when it happens, it's a major headache. The problem arises when either the compound or an impurity crystallizes in the column, forming a solid barrier that blocks solvent flow. The prognosis is not good- console yourself by noting the problem so you can avoid loading the loathsome mixture onto a normal column in the future. You will either need to employ some pre-chromatography purification technique or use a very wide column and a lot of silica.
*If your problem is not on this list, post it on Q & A!*