It depends on the class of compounds you are seaching. Oxidation, hydrolisis, etc., can occur, generating artefacts. Also, changes can occur during solvent evaporation. I use rota-vapor to evaporate solvent, but at low tempeture (less than 40 oC). At the end, to dry the fraction, the ideal is a flux of nitrogen. However, if you (like me) have not enough budget, a water bath, also at low temperature (up to 60 oC), can be used. However, take care with volatile compounds or those can suffer degradation under light and heat.

I think no, In column with solvent there is no oxidation occurs however after isolated a compound and left in exposure there may be a chance of oxidation.

It depends on the class of compounds you are seaching. Oxidation, hydrolisis, etc., can occur, generating artefacts. Also, changes can occur during solvent evaporation. I use rota-vapor to evaporate solvent, but at low tempeture (less than 40 oC). At the end, to dry the fraction, the ideal is a flux of nitrogen. However, if you (like me) have not enough budget, a water bath, also at low temperature (up to 60 oC), can be used. However, take care with volatile compounds or those can suffer degradation under light and heat.

Like Dr. Silveira said, compounds can oxidize or hydrolize during purification. Silica can be slightly acidic; one can try another media such as alumina or diol- derivitized columns (use diol like silica- it is less retentive so a weaker solvent system can be used). An amine- derivitized column can also be run as a normal phase column with the same solvent, but it is basic.

Can you explain us which kind of compound you're trying to purify?

Dear Wesley

I agreed with Jack and Silveira. Oxidation or degradation can occur during CC.It is hard to find out unless you are doing NMR fingerprint of extract before loading to the column. Some time even with NMR it is hard to find artifact, it is just depend on compound class and sample. I always prefer to treat silica with 10% NaHCO3 that neutralize or reduce the acidity of silica, so we can assume more safer isolation of compound. This is also time and climate dependent.

Essential oils are basically composed of two classes of compound, terpenes and phenylpropenes. Terpenes, depending on 5-carbon building block (isoprene unit), can be sub-divided into mono-, sesqui-, and di-terpenes in which the number of isoprene units are 2, 3 and 4, respectively. Further derivatives of terpenes occur due to the presence or absence of a ring structure, double bond, addition of oxygen or stereochemistry. It is estimated that there are more than 1000 monoterpenes and 3000 sesquiterpenes. On the other hand, phenylpropenes consist of a 6-carbon aromatic ring with a 3-carbon side chain attached (C6-C3 compounds). Unlike terpenes, only approximately 50 phenylpropenes are described.

It is hard to believe that isoprene unit can break on silica gel CC.

There is no chance for oxidation of compounds (dried as well as fresh plant extract) in column chromatography. For distillation process water bath and rotary evaporator is more convenient.. In case of essential oil normal column chromatography is not good, that case u choose flash chromatography

I think there is no chance for oxidation in the column chromatography. But I had encountered oxidation of my sample in the evaporation process using rotary evaporator. So I gave my sample to freeze-drying.

There is a chance for oxidation in the column but it depends on the kind of substance you are extracting, also the light could modify it, so I`d recommend you tell us about your compound to help properly.

Dear Servin

It is always good to have a strong reductant in your extraction, storage and elution solvents that can protect your compound from oxidation. The reason is dissolved oxygen in your solvent can sometimes oxidize your compound. However, the reductant should be soluble in the solvent you are using and should not interfere with the elution properties of your column. I have used the non polar, reducing compound BHT (Butyl Hydroxy Toluene) as a protective reductant during extraction, purification and TLC separation of poly unsaturated fatty acids. The compound is of low molecular weight, volatile and did not interfere with the mobility of fatty acids on silica matrix. it easily evaporated away when you dried the fatty acid down under a stream of nitrogen. If your compound is water soluble, you would probably use a similar non interfering water soluble reductant ( Ascorbate comes to mind, if that suits your requirements) in all your solutions.

Secondly rotavapor at low temperature and stream of nitrogen is probably the best for concentrating/drying down the compound.

Hope that helps

Hi,

The information you provided is limited and therefore these answers are somewhat hypothetical.

- If your compound is obtained by distillation then you probably should not use vacuum to remove solvent because you may lose some compound. Keep this is mind if using rotavapor or freeze-drying.

- Yes, NO DOUBT some compounds will degrade during column chromatography or distillation.

- Answers above contain a lot of good advice, I can only add these tips out of personal experience:

1) take care if your column is exposed to strong light and/or high room temperature;

2) some plant extracts do contain pro-oxidants (metals, etc) so sometimes adding a little EDTA may help;

3) the solvents you are using may be introducing contaminants/pro-oxidants, and your water is sometimes the solvent you least suspect but the very worse of them!

Hope it helps.

Can you explain us which kind of compound you're trying to purify? Maybe we can help you

I think the chances of oxidation in the column are minimal, compared with the chances of oxidation before you use the column. However, once the compound has been isolated, the posibility of oxidation increases, because the isolated compound would be whithout other compounds in the mixture that were acting as antioxidants,

And for your second question I think it depends on differences of boiling points of the compound you are trying to isolate and the solvent. If you use a low boiling point solvent you wouldn't have troubles.

The result depends on the compound that you isolated. As well as I known, most of the natural compound are stable except a few compound, such as tannins.

I think the chance of oxidation of phytochemicals in ordinary conditions such as room temperature or distillation at optimum boiling range of the solvent in which u had eluted that compound. But chances of photooxidation of light sensitive compounds which are very minimal in plants. So it is adviced to use rotary evaporator through out the isolation procedure unless you are confident that distillation would retain your compound intact.

It depends of the chemical nature of phytocompound,, but if it is a polyphenol, the possibility for any degrae of oxidation is high.

All above Scientists have gave very good suggestions to prevent oxidation in column chromatography. If the solvent used for eluting compounds is non polar or do not contain water or ethanol or any polar solvent which has great affinity toward oxygen, the chance of oxidation is rare. if the compound is in solvent free from oxygen, no oxidation is possible. Therefore combine all fractions of column, concentrate under reduced pressure, at low temperature and in inert atmosphere will prevent oxidation. i will not suggest for addition of any compound for preventing oxidation.,

I have experieced oxidation in work up procedures a few times, it all depends on the nature and the relative sensitivity of the oxidable moiety, for istance a free cysteine in a protein can be preserved for long time in its reduces form just adding metal scavangers (metallic ions catalyze oxidation) as EDTA . The slight acidity of normal silicagel sometimes can do bad trick on sensitive eluted compounds too.

Very thanks to all for your valuable answers.....

Thank you Dr. Lohar sir, i took low polar solvent in column packed with ethyl acetate slurry (activated silica with ethyl acetate extract- dry)..the fractions was heated using water bath, condensed and distilled and i got a single compound finally.. i stored it in a glass beaker...is there any chance of oxidation of that compound....

It's rare, but if it occurs, it depend largely on the type or nature of phytocompounds involved as well as their relative stability. The presence of impurity in the solvent system may encourage oxidation, however, this may be removed by making sure that the solvents to be used in running the columns are distilled prior to use. The column should also be run within the shortest time to limit its exposure to atmospheric oxygen.

Dear Dr Servin, It is fine that you got a single pure compound, but do not keep it open in beaker, Dry it immediately, under nitrogen atmosphere, and store in sealed amber colored vial to avoid contact with air/oxygen humidity and light. There are the three factors influence oxidation.

Okay sir thank you...

Why not? It depends on the nature of your compound. Try to run your column under nitrogen pressure (not just air) and degas (deaereate) your solvents; this will diminish the severity of your problem.

Sir, I recommend to you the HSCCC (High speed countercurrent chromatography) technique. It is very safe, very fast and economic in solvents. Actually, this technique is very safe to polar compounds. I never had any kind of problem in phytocompounds oxidation.