*This post is part of a series of posts based on my research as a masters student at UGA. To find out more go to my page Holy Basil Research.

Like I said in yesterday’s post on building protocols for essential oil distillation, once we distilled the essential oil, we had to figure out how to get it out of the clevenger trap and get an accurate mass to know how much essential oil is in a specific amount of plant material.

There were several challenges in answering this question. First of all, before we can collect the oil for analysis we have to filter through a compound called Anhydrous Sodium Sulfate.

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It binds to all the water molecules but allows the pure essential oil to flow through. This is an important step because water in the sample will contaminate our results when we run it through the Gas Chromatograph Mass Spectrometer to identify the compounds in our essential oil. We brainstormed different ways to do this. First we thought of a filter and funnel, but thought we would lose too much essential oil that way.

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We decided to roll up some glass wool and put it into a disposable glass pipette. The glass wool acts as a barrier to keep the sodium sulfate from washing or falling through the tip of the pipette. Then we added the sodium sulfate over the glass wool. Now we could filter essential oil through the pipette and into a container.

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This is glass wool (it looks just like cotton candy!). But you don’t want to eat it and you have to use gloves to handle it or it will tear apart your hands.

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Filtering the oil through the sodium sulfate and glass wool worked great. The next problem came from a fraction of the oil getting stuck in the glass wool. We had to find a way to wash the oil through without getting any water into our sample to get an accurate mass of essential oil from our distillation. This is usually done with hexane since it is non-polar and doesn’t react with water. The problem is that we can’t have any hexane in our sample that we use for analysis. It needs to be pure essential oil. I consulted with countless people in the best way to do this. I talked to food scientists, engineers, horticulturalists, chemists, people in the industry, etc… Each of them helped me a little bit and I came up with a plan.

To begin, we needed to figure out the accuracy of our collection method. So, I purchased holy basil essential oil and we measured out 1 ml and recorded the mass.

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Then we pipetted it into the clevenger trap just like it would be at the end of a distillation.

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Since we have a known quantity at the beginning, we could compare that to the final quantity to determine our percent recovery and the effectiveness of our collection method.

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We used the valve at the bottom of the clevenger to drain off as much water as possible. Then we set up the glass pipette with glass wool and sodium sulfate under the tip of the clevenger trap to let the essential oil flow through the pipette and into the amber bottle at the bottom.

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Once the majority of the essential oil was finished flowing through the pipette we removed the amber bottle. It contained the pure essential oil that we will use for analysis. The fraction of essential oil that was stuck in the glass wool we collected as a second fraction by putting hexane through it.

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We figured we could measure out 1 mL of hexane, record the mass, and pipette it into the glass pipette and collect the hexane and second fraction of essential oil in a beaker.

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Then we would just subtract the mass of the hexane and the mass of the beaker to get the mass of the second fraction of essential oil. Good plan, right?

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This was a great idea until we realized how quickly hexane evaporates! It is highly volatile and even though we worked quickly, it really skewed our results.

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We also had a problem with small amounts of hexane and essential oil getting stuck in the tip of the clevenger trap with no way to blow them through. This also negatively affected our percent recovery. Also, the tip of the clevenger trap was fairly large so we frequently had a problem with essential oil spilling over the side of the glass pipette which was bad and very frustrating. When we tried to fit the opening of the glass pipette over the tip of the clevenger trap a suction would be created and the essential oil wouldn’t drain. When we separated them to relieve the suction then essential oil would spill everywhere and we would have to start over.

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We worked a whole day and ran this experiment 8 times getting a percent recovery from 65% -85%. For a good protocol we were shooting for a 5% range. This was not good enough.

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We gathered our papers, took notes on each experiment and we kept making changes to the protocol.

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First we figured out our scale was faulty. So we started using a better scale.

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Then we decided to pipette the oil from the top of the water instead of letting it flow through the tip at the bottom so we didn’t lose any oil there.

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Finally, we decided to cover the top of the beaker with parafilm for collecting the second fraction of essential oil and hexane to prevent the hexane from evaporating so quickly.

By our 10th try we got to a 91.6% recovery. Once we felt like we had a good protocol, we had to repeat it 3 times the exact same way to get a range. After 3 times, we were able to get a 89.7-91.6% recovery range. We were very pleased. Now we know that we can base our results on ~90% recovery rate with a standard error of +/- 2%.

This was a big challenge and I am so happy that we spent the time to create a good method for distilling and collecting the essential oil. Tomorrow I will walk you through the whole process now that all the protocols have been built and we can start running distillations with real samples from my research!

If you are interested in reading more about my funding, research and masters program, you can check out my page Holy Basil Research!