Extracting high-purity asarone from calamus oil

[00:00:00]Have a look at this molecule, 2,4,5-trimethoxybenzaldehyde.

[00:00:04]It's a research chemical that's pretty hard to make from scratch. If you want to prepare a molecule like this, it's usually best to start from a more widely available molecule that has most of the structural features already in place.

[00:00:15]The most similar molecule that's readily available is vanillin, which has two of the three oxygens already on the ring.

[00:00:20]But, the biggest challenge there is selectively getting a substituent onto the two position instead of the three or the six.

[00:00:27]Although the five hydroxy group activates the three position, the five methoxy group activates two and six positions, meaning that any kind of substitution reaction on the ring would result in a mess of three different isomers, two of which would be unwanted.

[00:00:40]There is, however, a molecule that occurs in nature that already has a 2,4,5-trimethoxybenzene ring. This molecule is asarone, the major component in the essential oil of Acorus calamus.

[00:00:50]This plant, known among many other names as the sweet flag, the calamus root, or the muskrat root, grows in Asia, Europe, and North America, and the percentage of asarone is high in the Indian variants.

[00:01:02]Asarone exists in three different isomers, alpha, beta, and gamma, depending on the position and orientation of the double bond.

[00:01:08]The main one found in calamus oil is beta asarone, which has the double bond next to the ring in a cis orientation.

[00:01:14]Incidentally, the name asarone is misleading as it implies the molecule has a ketone group, but it doesn't.

[00:01:20]Although the alpha isomer has some medicinal benefits, the beta isomer is actually pretty nasty stuff, being a suspected human carcinogen.

[00:01:27]There are several ways to isolate asarone from calamus oil. The most common method is high-temperature vacuum distillation, but calamus oil is not cheap or easy to find. My apparatus is too big to deal with such small quantities of material, and I don't have enough confidence to heat my glassware to nearly 200° under vacuum. I've done this once before, and although the apparatus didn't implode, the round-bottom flask containing distillate got a nice new concave base like a fancy wine bottle. In fairness, this was due to me heating it pretty aggressively.

[00:01:56]Another method is to isomerize beta-asarone to alpha-asarone, which is a crystalline solid and separate it from the rest of the oil, which also contains terpenes, fatty acids, and other unwanted compounds.

[00:02:06]Olefins next to aromatic systems can be isomerized with catalytic amounts of iodine in the presence of in the presence of heat or UV lights, and the reaction is faster with the electron-rich aromatic systems. Since the alpha and beta isomers of asarone both contain three methoxy groups, they have very electron-rich aromatic systems, making this a viable method.

[00:02:25]The reagents used were calamus oil, 10 g, ethyl acetate, 40 ml, iodine, a tiny unmeasurable trace, sodium thiosulfate, 2 g, sodium hydroxide, 4 g, deionized water, 60 g, sodium sulfate, heptane, 25 g, and methanol, 20 g.

[00:02:48]Ethyl acetate was weighed into a round-bottom flask. Calamus oil and iodine were dissolved in it, then the mixture was refluxed for an hour and a half.

[00:02:56]As the mixture was cooling, sodium thiosulfate was made up to 10% with water, and sodium hydroxide was also made up to 10% with water.

[00:03:04]Once it was cooled, the mixture was washed with the sodium thiosulfate solution to neutralize the iodine, and then with the sodium hydroxide solution to remove any fatty acids or molecules with phenol groups.

[00:03:14]Given that the caustic soda solution turned orange and a pasty soap-like precipitate formed in it soon after, it seems that these materials formed a significant part of the oil.

[00:03:23]The now turbid reaction mixture was filtered by vacuum, dried with sodium sulfate, then filtered again by gravity.

[00:03:28]Gravity filtration was slowed down by formation of a white crystalline precipitate, which may well have been the desired product.

[00:03:35]This also appeared on the filter paper once the sodium sulfate had been discarded and it had dried out.

[00:03:40]This had not happened in previous runs where I'd used less caustic soda. Once washed, the solvent was removed from the vacuum mixture by vacuum distillation to limit decomposition and allow as much of the solvent to be boiled off as possible.

[00:03:52]It yielded a viscous oily liquid with suspended particles in it. Again, different to earlier runs which had just produced viscous oil. This mixture was suspended in heptane, heated to boiling then filtered by gravity to remove any insoluble material.

[00:04:06]The insoluble material in question was some brown gunk that stuck to the flask.

[00:04:10]Once filtered, the filtrate was reheated to 80° boiled down to about half its initial volume then cooled to room temperature with stirring where it became a viscous clear liquid. It was then cooled in the fridge overnight. The reason for the stirring during cool down is that all forms of asarone have melting points below the boiling point of heptane and I found that alpha asarone tends to form super saturated solutions.

[00:04:30]Its apparent failure to precipitate was a major cause of poor yields and general frustration in my earlier attempts to isolate it.

[00:04:37]Cooling in the fridge caused solid material to form. Methanol was added to the mixture causing my white solids to appear.

[00:04:43]The mixture was heated to boiling and the volume was reduced to around 20 mil and the mixture was cooled to room temperature with stirring with the amount of placed in the beaker to generate turbulence, help with nucleation and help the solution break free from the tyranny of super saturation.

[00:04:58]Around 45° fine off-white solids formed in the mixture and within seconds a substantial amount of product precipitated out. It was cooled in the fridge overnight. The mixture was placed on the pump but there was so little liquid left that nothing came through.

[00:05:10]It was washed out twice with methanol to remove entrained liquid and dried on the pump for a further 15 minutes. The final product, alpha asarone, was isolated as a white powder with a melting point of 62 to 66° with a balsamic slightly woody smell similar to that of calamus oil itself. The yield was 5.2 g. This is an efficient and slightly laborious method of producing high purity alpha asarone.

[00:05:33]Without knowing how much beta asarone there was in the starting material, I can't give an exact figure of how efficient it is.

[00:05:38]As pure beta asarone solidifies around 15 to 20°, and my calamus oil stayed liquid at 4°, it's fair to say it contained a significant amount of non-ascarole material.

[00:05:48]The way the reaction works is this. At high temperatures, iodine molecules split into iodine radicals. The most obvious sign being the fact the orange color characteristic of iodine fades as the mixture heats up.

[00:05:59]An iodine radical attacks the double bond of the ascarole molecule forming a carbon-centered radical.

[00:06:04]As carbon is a much smaller, less polarizable atom than iodine, this radical is less stable than the iodine radical and it mostly reverts back to the starting material reforming the double bond.

[00:06:13]However, the more thermodynamically favored double bond, the trans bond, is formed by preference. The more electron-rich the aromatic system is, the more reactive the double bond is to electrophiles like radicals, and the faster the reaction is.

[00:06:25]As the cis and trans isomers are in equilibrium, 100% conversion is generally not achievable, but it doesn't show up the trans isomer as the major product. I've longer reaction times in previous runs than these do not result in greater yields.

[00:06:38]The reaction can be carried out with catalytic quantities of iodine as in less than 1 mol % so any addition products are kept to a minimum.

[00:06:45]After that, extracting the ascarole from the mixture is a process of elimination, first removing the iodine from the mixture with sodium thiosulfate, then the phenols and fatty acids with sodium hydroxide.

[00:06:55]Ascarole is insoluble in heptane at room temperature but dissolves at high temperatures. Anything insoluble in hot heptane is not the desired product so it can be removed by filtration.

[00:07:04]In a similar way, it's insoluble in methanol at low temperatures due to its low dipole moment and anything remaining that is soluble in methanol can be removed by filtration.

[00:07:13]The fairly sharp melting point indicates any impurities present are insignificant.

[00:07:18]All in all, this is a reasonably efficient and quite enjoyable way to extract a white powder from a yellow liquid. In the future, I can hopefully demonstrate a way to convert ascarole into the benzaldehyde shown at the start.

[00:07:28]I hope you enjoyed this video. Thank you for watching.