Science Buddies: "Ask an Expert"

Hi Sybee,
I now realize I was not very clear about what I was asking. My teacher was wondering if could put the liquid chlorophyll in the steamer and get the phytol out that way. Sorry about the confusion. I was also wondering if this would work as a steamer because it is my only glass bowl and this is the pot it fit in best [photo removed by student].

-Vlance

Oh, I should have realized that's what you meant. You already had the chlorophyll extract so just go ahead and steam that until it turns brown (hopefully it will!).

Your steamer set-up looks fine. I'm debating whether or not to put a lid over the bowl. What do you think? How much chlorophyll extract plus vinegar do you have? I just don't want to have all the water evaporate; but, on the other hand, maybe evaporating some of the water would be a good idea to make the extract more concentrated.

Did you use spinach leaves to make the chlorophyll extract? How much leaves did you use? How much phytol are you supposed to use to clean up an oil spill of a certain area? Will you have enough to do the experiment at least three times? I'm trying to think of any potential problems now before you get too far along. Please, if you think of something or have any questions--now is the time to bring them up.

For a control you could add vinegar to water in the same proportions as for your phytol preparation but without adding any chlorophyll. Then you would neutralize the vinegar with baking soda and use that solution as the control.

What is your deadline for finishing this project? You still have a lot to do.

I think that's all. Please answer my questions so I have that information. Good luck getting the chlorophyll to release its phytol.

Sybee

Hi Sybee,
I think that I will leave the lid to the steamer off so that the phytol will be more concentrated. I bought the liquid chlorophyll at the vitamin shop already extracted for when I was trying the vinegar without heat. I realized that I would most likely not get very much phytol when I tried to separate it from the chlorophyll so I will be doing micro chemistry. I am going to combine the phytol with olive oil to try and align the phytol molecules better so it can break the surface tension and congeal the oil better. I have lots of liquid chlorophyll so I can do many tests with it. I need to have my data in by March 4th. If we can't figure out how to get the phytol out by the middle of February my parents said that we can buy some already extracted phytol but I would like to extract it myself.
-Vlance

Hmm...
And here I thought you were cooking spinach leaves!

It is just my opinion without evidence, but I think fresh chlorophyll would be the best choice for an experiment. What do you think? The procedure for isolating chlorophyll from spinach leaves is pretty easy. Can you find out how the liquid chlorophyll you bought was prepared? It may have some chemical stabilizers added to it that make it more difficult to break the bond to release the phytol. Let me know what you find out about the liquid chlorophyll and whether you want to try making the chlorophyll extract yourself.

Now, that we've spent all this time figuring out how to make phytol, I did a search for where to buy it and found a company that sells it--2 ml for $13

https://trueterpenes.com/products/terpenes/phytol/

I still think preparing it yourself from fresh spinach leaves is the most satisfying way to do it, and it may turn out to be the most active phytol. You can try the liquid chlorophyll, but if you heat it with vinegar on the steamer and it never turns brown then it may have been modified in some way to prevent the phytol from being broken off by acid.

I hope whatever you decide to do works!

Sybee

I think that the easiest thing for me to do is try with the liquid chlorophyll first and if that doesn’t work I will try to get the Phytol out of the spinach. I will try using the steamer method on the liquid chlorophyll tomorrow and let you know what happens. Do you know what the maximum amount of time I should simmer the liquid chlorophyll trying to get the Phytol out for before I try the spinach?

-Vlance

I really have no idea. When I cook green beans or kale I only steam them for 3-4 minutes in the microwave on high, so I would estimate that it takes maybe 20 minutes on fairly high heat to make them turn brownish green, but that is without acid present.

How much vinegar are you adding to how much chlorophyll? Vinegar is a weak acid and is not going to break the phytol bond as well as a strong acid like hydrochloric, which is what you would use in the lab.

Remember to take before and after pictures of the chlorophyll. Hope it works!

Sybee

Hi Sybee,
Today my mom had a question about my experiment and I didn't know the answer. She once put a glass bowl in a pot of boiling water and it exploded, will that happen when I put the bowl in the simmering water? Also she was wondering if the judges would question my project and think it was more about getting the phytol out than creating a oil spill removal spray? I thought no because this is me getting the phytol out to start the project but I wanted to ask your opinion.

-Vlance

Hi VL,

I am so glad that you are asking questions! That's the mark of a scientist.

A glass bowl could break if it was cold and immediately immersed in boiling water. Likewise, a hot bowl put into cold water could shatter. Pyres glass like the Fire King or Anchor Hocking brands is more resistant to thermal stress.

What I would do is is put a couple inches of water in the bottom of the pot, put the glass bowl on it and heat the water. That way the bowl will be heating at the same rate and won't crack. You can also put the chlorophyll solution in the bowl so that it can start heating.

Your project asks the question, Does using phytol improve the removal of oil from an oil spill on water? The phytol is the key part of your project, but it is not the object of it. When you write up the results you don't need to spend a lot of time describing the phytol preparation. Just show a diagram of the hydrolysis reaction of the chlorophyll molecule with acid and heat to produce phytol plus the remnant of the chlorophyll (phaeophorbide)--http://www.ch.ic.ac.uk/local/projects/steer/chloro.htm

Take note that we are making an assumption here that the mixture of phytol and phaeophorbide that you prepare will promote oil recovery exactly the same as phytol alone. I don't know whether that is true, but I am betting that it is. You could go ahead and order the phytol from the source I sent you so that you could test it along with your phytol prep. Testing two different compounds will strengthen your results and make your project A++.

Hurry up and make the phytol! I want to know how well this hydrolysis method works.

Best wishes,

Sybee

Hi Sybee,
I wanted to update you on my project. I currently have the chlorophyll mixed with vinegar in the "steamer" at medium heat and the chlorophyll is moving around. I'm not sure what is happening. This a video of what I was seeing. I hope you can tell me whats happening [removed by student].

-Vlance

Hi Sybee,
I think that I need to purchase the phytol. I tried heating up the chlorophyll will the vinegar mixed with it but nothing happened. I gave it a couple of hours but my stove started making abnormal noises when the "steamer" was on it. Thank you so much for you advice I will make sure to tell the judges all of the things I tried. For my science fair board I could use your name and talk about how all the ways you helped me?

Thanks for all the help,
Vlance

Hi VL,

Thanks for updating me on your progress. That's the world of real science, unfortunately, where things often don't work the way you expect. I'm pretty sure the problem is that you are not able to use an acid at the strength needed to break the phytol bond. You would probably need to use hydrochloric acid at a concentration of 15-20%.

If you heated a bunch of spinach leaves with some salad oil in a pan to stir-fry temperatures (325 F or so) I think you could achieve breakage of phytol from the chlorophyll. That is what i do when I cook greens like chard and Napa cabbage and they always turn a little brown if i heat them too long.

Well, go ahead and order the phytol. I don't know how much you will need but 1-2 ounces of pure phytol should do it. The next challenge will be measuring the exact amount of oil recovered from the clean-up of the 'spill' and the sooner you get started on that the better.

Thank you for wishing to acknowledge me personally. It is my pleasure to try and help you do your project and to understand how the scientific method works. The U.S. needs more good, young scientists and you might be the one to discover a great new process or invention. Scientific research and engineering are great careers--challenging, satisfying and exciting. You can say that you received help and mentoring from the experts at Scibuddies and that you highly recommend us. I am already looking forward to working with you on your next science project!

Good luck,

Sybee

Hi Sybee,
I know that I said that I was just gonna buy the phytol but my teacher just emailed me and told me she had sulfuric acid. I will give that a try some time next week to try and get the phytol out but if I can't then I will buy the already extracted phytol. I will update you if this will allow me to get the phytol out. I will also be heating the chlorophyll at the same time just not in a "steamer".

-Vlance

Hi V,

Hmm...
Sulfuric acid would not be my first choice. Doesn't she have or can get hydrochloric acid (HCl)? Sulfuric acid is a very strong oxidizer and I would be concerned that it might destroy part of the phytol molecule.

Ask your teacher if she has HCl. If not it can usually be purchased from a place that sells pool supplies or from a home or hardware store. Just tell her to ask for muriatic acid, the common name for hydrochloric acid. The concentration is usually about 32% HCl. Be careful with this! Your teacher should know how to handle acids safely. You must wear goggles or face shield, nitrile gloves and a lab coat as personal protection. The acid should be used in a fume hood because its vapors are very irritating.

I will try to find out what concentration of HCl you should use for hydrolysis of chlorophyll. I still think you should make a fresh preparation of chlorophyll from spinach leaves. I'm concerned that the chlorophyll preparation that you are using may have a chemical stabilizer added to it that prevents breakage of the phytol bond. Just do a search on Google or Youtube for how to prepare spinach chlorophyll. This is a common lab exercise and fairly simple to do.

Sybee

Hi VL,

I was wondering what you had decided to do for producing the phytol you need. I did another search for acid hydrolysis of chlorophyll and did find out that sulfuric acid can be used for hydrolysis:

https://pubs.acs.org/doi/pdf/10.1021/ic00134a056

I tried to get this 1982 article through my university library but could not find the citation. I don't think their databases go back that far for this journal.

If there is a method for acid hydrolysis of chlorophyll on the web, I don't know where it is hiding. I could not find it!

What I would do is make a range of dilutions of acid in your chlorophyll solution and record what happens. Just be careful working with strong acids. ALWAYS where eye protection, gloves and a lab coat or full apron to protect your clothing. Work with your teacher or someone familiar with chemistry in your school lab.

Let me know what you decide to do.

Sybee

Hi Sybee,
I am very sorry for not updating you sooner about my project. I had to give up on trying to get the phytol out myself, so I just bought about 8 ml for my project. So far it looks like I will be actually performing the experiment this Wednesday. Last night I was doing somemore research about my project and I saw that the phytol was going to reat with a cation (salt in the salt water) and I did not need the olive oil. I reaized this caused a problem beause I do not have enough pure phytol to perform this experiment and I have confirmed this as a engineering project. I was thinking that I could just use fresh water to fill in what I was going to use the olive oil for each prototype. I found out that phytol was actually hazardous so I will be using a fume hood but I thought that the water might actually help dilute the phytol so it will be less hazardous but still work. Do you think this will work, and if not what could I use with the phytol instead?

-Vlance

Hi Vlance.

I wish your phytol extraction had worked. That would have added a DIY aspect to the project. But I'm sure you learned some things about running experiments in a lab. Many times I have had to modify a procedure because some part of it did not work the way I expected it to. The important thing is to think through how the change might affect your experiment and you did that.

Can you give me some more details about what you read. I don't think I understood what you were saying. Did you mean that you could not do the oil clean-up experiment in salt water because the sodium cation reacts with phytol? Where did you read that and what exactly did it say?

You said "...I did not need the olive oil". What did you mean? How can you do the project without oil??

How do you know that you don't have enough phytol?

You said "...I have confirmed this as an engineering project." OK. What's wrong with that? Engineers and chemists and biologists all work together to solve problems in the environment. They have to. Chemists have to develop the compounds to dissolve or break up the oil, biologists need to check them for toxicity and engineers have to figure out how to collect the oil spill. Can you please explain in more detail exactly what you meant. Accurate communication is VERY important in science.

Of course you can use fresh water for your experiments. Oil spills can happen anywhere.

How do you know that phytol is toxic? It is part of the chlorophyll molecule. We eat it every time we eat green plants. Did you mean that the phytol you purchased contains a solvent that might be toxic? Please explain better. Working in a fume hood is necessary if you are using certain organic chemicals. It depends on the chemical. More information, please!

If you are having a problem with your project, don't wait--talk to us! We are here especially to help you when you hit a snag. Tell me what's going on and I can maybe suggest a work-around.

Sybee

Hi Sybee,
Yesterday I performed part of my science fair project. Sadly I came out unsuccessful and the phytol mixed with avocado oil did not congeal or group the motor oil I put on salt water. I was very disappointed but then one of my friends found a article about my project and it says that it takes a couple of hours for the phytol to work! The article also said that for the phytol to work it had to be combined with an amino acid. I understand that amino acids are related to protein so does that mean that the avocado oil is high in amino acids? I have some of the leftover solution from my first 2 prototypes so far and will leave them on the oil for longer to try again but I'm not sure if I have the right ingredients now. This is a link to the article; https://www.popularmechanics.com/scienc ... odegrades/

-Vlance

Hi Vlance,

I read the article from Pop Mechanics July 2015 about using phytol as a biodegradable 'oil herder'. Now I understand better how the phytol is supposed to work.

Have you had a course in basic chemistry yet? If you look at a representation of the phytol molecule (https://en.wikipedia.org/wiki/Phytol) you can see that one end has an --OH group on it. That is why it is called an alcohol. It is also why that part of the phytol molecule is soluble in water (either salt or fresh, doesn't matter). Forget about the amino acid. I don't know why the article mentioned that. The --OH group is all that is needed.

The other part of the phytol molecule where you see the 'sawtooth' effect is the di-terpene part. That part is soluble in oil.

So, the reason that phytol is able to reduce the surface tension of water and cause oil molecules to clump together is because it has both a water-soluble and an oil-soluble part.

Have you had enough chemistry to follow what I am saying? Please let me know if you don't understand something.

There are still a lot of things that you haven't explained to me. I can't help very well if you won't take the time to answer my questions. Why did you mix phytol with avocado oil? I read an article in ScienceAdvances (https://www.ncbi.nlm.nih.gov/pmc/articl ... figure/F1/) that shows that the phytol was simply sprayed around the oil slick, which then contracted into a smaller mass that can be burned. How much phytol did you use? How much motor oil? How large was the surface area of the water that you put the oil on? How did you apply the phytol to the oil slick?

I think mixing the phytol with avocado oil prevented it from working properly. As I understand the method, you should spray phytol solution AROUND the oil slick and then measure how much the oil slick contracts. What I don't know and what you need to determine by experiment is how much phytol is needed for a given amount of oil. This is something that oil spill workers would absolutely have to know. I suspect that what they do is spray phytol around the oil slick and watch what happens, then spray some more until the oil has contracted to the point where they can burn all of it.

How much phytol do you have left? I would try the oil slick again using just a small amount of motor oil and straight phytol, NOT mixed with oil. Put a small drop of the motor oil in the center of the water and try to measure the diameter of the slick just before you apply the phytol. I don't know what would be the best way to get the phytol solution around the oil. I guess you could spray it, but the sprayer would have to be pretty small because the volume of phytol is small. How did you apply it before?

Sybee

Hi Sybee,
I have not really done any basic chemistry yet but I think I'm following what your saying. I added the avocado oil to the phytol because one article told me a plant based fat was needed to break the surface tension and I was trying to make the phytol less toxic to marine life because it came with that warning on the bottle. Why would the avocado oil have stopped the phytol from working? I have 5 milliliters of phytol left so I need to be careful about what I do with it. I was putting the motor oil and phytol on salt water from the ocean with a pipette. This is a link to the actual study of the experiment that my teacher recently found, I'm not fully understanding all the science terminology so I was wondering if you could help me understand what parts are important to the experiment, [link no longer valid]

Thank you,
Vlance

Oh, so chemistry is sort of a black box for you right now. OK, I'll try to explain in language you can understand. If not, make a note of your questions and send them and I will answer them one by one. Chemistry is important stuff and I want you to have an accurate idea of molecules and reactions.

Phytol is a molecule as I said, and one end can dissolve in water while the other end dissolves in oil. Maybe that doesn't make any sense to you, but someday it will. When you added oil to the phytol the oil end was saturated with avocado oil and was not free to interact with the motor oil. The change in surface tension can only occur when the water end of phytol is in the water and the oil end is in motor oil.

Don't worry about toxicity to marine life at this point. All you want to do now is show that phytol can cause an oil slick to contract and make it easier to collect or burn the oil. Leave the chemistry to the company that is trying to create a nontoxic, biodegradable form of phytol for oil slick remediation.

You still have not told me how much motor oil you put onto what surface area of water. Maybe you should make a list of my questions and answer them one by one. Your answers are necessary for me to think about the experiment and give you good advice.

How large are the bowls [the diameter is what i am after] that you will be using to test the phytol? You need to use the exact same bowl and the same amount of water for each test. After you have decided on the amount of water to use--maybe to within a half inch of the rim of the bowl--measure the diameter of the water and use the formula for the area of a circle to calculate the surface area of the water. This is kept constant and it is a number that you must report as part of your project.

You want to add enough motor oil to create a slick but not cover the entire water surface. That is why i keep asking you how much oil you are adding to how much water. You have to be able to say how much you used, so you need to know the volume of one drop of oil if you are using a dropper. You can measure this but you will need a cylinder graduated in milliliters that you can fill to the mark while counting drops.

The next problem then is how to apply the phytol. Remember, the phytol is sprayed AROUND the slick, not ON the slick. You don't want to break up the slick, you want it to contract into a smaller area that can be collected more easily. I really have no idea how much phytol you need to add for a certain amount of oil. How are you applying the phytol? I think spraying would be too difficult so you have to use a small dropper to put it around the slick. This is going to be hard because you have to measure the diameter of the slick before you add the phytol and then after a certain length of time when the slick has contracted as far as it will.

There was a server error when i tried to look at the email link you sent. I will try again later.

Read my explanations and suggestions carefully, please answer ALL my questions and let's try to decide the best way for you to proceed.

Sybee

Hi Sybee,
When I conducted my experiment I used 1.5 milliliters of ocean water, with 1/2 a milliliter of motor oil on it, and 1/4 milliliter of the phytol and avocado oil on that. I am using a paint pallet (the on in this picture [photo removed by student) and the diameter of each circle is 3.5 centimeters. I was applying the phytol with a pipette but the liquid was a little thicker than I expected (possible because of the avocado oil) so it was squirting on top of oil. I found that the oil would spread out around the circle of the paint pallets. This is the same article I was telling you about that you couldn't access only I tried sending it to you in a different way, http://advances.sciencemag.org/content/ ... 65/tab-pdf

-Vlance

I read the methods part of the ScienceAdvances paper and it leaves out some important details, like the size of the plastic tray that they used to make the oil slick in:

"A plastic tray was filled with water up to 2 cm deep, and then the calculated amount of oil was poured on the water surface and allowed to spread to equilibrium to create a thin film of oil. A digital photograph was taken from an automatic overhead camera for subsequent oil area analysis. Next, the recommended dose of green herder (20 microliters of herder’s stock solution in toluene delivering 2.34 mg of herder based on the standard dose of 150 mg/m2) was supplied using a micropipette and, instantaneously, the thin oil layer contracted to a thick oil slick."

Looking at the photo in Fig. 3b it is not possible to tell how large the trays are, but I'm sure they are larger than 3.5 cm across. I can see some clear areas around the oil slick.

In order to do this experiment you need to make an oil slick. That is a THIN film of oil that floats on the water but does not cover the entire surface.

Get some glass bowls all the same diameter--say 10 to 15 cm--and fill them to within 2.5 cm of the top with water. Now pipet a small amount of oil, say 100 ul (microliters) on top of the water in the center and watch what happens. The oil should spread out into a thin film and then stop with some clear areas around it. THAT is an oil slick. If 100 ul is too much oil use less; if not enough use more.

Now using the micropipette, pipet 100 ul of phytol only (NO OIL!) onto the water around the oil slick and watch what happens. I don't know how much phytol you will need to add so you will just have to add a small amount and see what happens. The main thing is to put the phytol on the water around the motor oil--not on top of it. As you see in the photo in Fig. 3c and d, the oil contracted into a smaller thicker area. That is what you are looking to happen.

Try this experiment and let me know what happened.

Sybee

Hi Sybee,
I am going to be testing out my experiment with your suggestions but I do not have the glass bowl you said I needed but I was wondering if plastic container would work because I have a lot with the exact diameter you said I needed.

-Vlance

Plastic containers should work OK. Try a little experiment first. Fill some bowls with water at a temp of around 20 C. You should measure the temp of the water and record that in your lab notebook.

Add one drop of crude oil to the center of the water in one bowl and measure how far the oil slick extends after five minutes, or so. The oil should not reach the edges of the bowl. If one drop is not enough, try two or three as needed. As long as you have a clear area around the slick to add the phytol the experiment will work.

Good luck! Let me know what happens with your little experiment.

Sybee

Hi Sybee,
I have some great news!!! Thanks to your tip on getting the oil spill to actually be a simulated oil spill, all of my prototypes worked (even the avocado ones). My next step is to test the toxicity of each prototype and evaluate which one I think is best.

Thank you so much,
Vlance

Yaay! Great news that your experiments are finally working. That was a wonderful idea you had to test a natural product like phytol to make it easier to clean up oil spills. Phytol should not be toxic to marine life since it breaks down quickly in the environment. Also, it is cheap and abundant!

When you have more questions about the science or in understanding journal articles for your write-up of the project, don't wait--ask NOW. We're always here to help give you a hand up to make your project the best it can be.

Sybee

Hi Sybee,
So as you know I tested the phytol and it worked but I couldn't not observe if the phytol was breaking down because it is a clear liquid. I was going to do another 2 trials and take the pH everyday until it gets back to a normal water pH. I also wanted to test how toxic the liquid was because I wanted to know if the phytol would harm marine life (on the website of the place it came from it said it was harmful to aquatic animals) but I don't know if doing these 2 sub tests would be like doing 2 more different science fair projects, but in my project proposal I said that I wanted the solution to biodegrade and be non-toxic. I want to know if I am packing too much into one science fair project because I want to make sure I don't confuse people about what my topic was.

-Vlance

Hi Vlance,

Congratulations on showing the efficacy of phytol in oil clean-up. I think that is enough for one school science project. Your plans to test for toxicity to marine life and the biodegradability of phytol are good and necessary. An environmental scientist would perform those tests as part of their project, but they have more time, people and money to do it.

Are you interested in marine biology/ecology? Do you live near the ocean or bay? If so you can continue your experiments by collecting some marine organisms like krill, plankton or cyanobacteria, exposing them to phytol and measuring the effect on survival, growth or behavior. Here are some websites with information on this:

https://en.wikipedia.org/wiki/Aquatic_toxicology
https://oehha.ca.gov/media/downloads/ec ... netox3.pdf
https://www.epa.gov/sites/production/fi ... l_2002.pdf
https://ehemj.com/article-1-48-en.pdf
https://www.sigmaaldrich.com/catalog/se ... xtEALw_wcB

I really don't know anything about the biodegradation of phytol in the ocean. There would be lots of factors that affect the breakdown--temperature, sunlight, pH, type of marine organisms present in the water, how much phytol was present and the effects of residual oil. Studying phytol breakdown in the ocean would take a lot of work and knowledge of chemistry, biochemistry and marine ecosystems. I hope you decide to become a scientist. It is a very satisfying career and offers a lifetime of exciting exploration and finding new things in our amazing natural world.

I hope you continue to use Scibuddies so we can work together again in the future.

Good luck!

Sybee

Hi Sybee,
Thank you for all your help! I am going to test the toxicity of the phytol on Daphnia, although they will be in fresh water it will still reflect the toxicity of the phytol. To see when the phytol breaks down I will take the ph of the water right after I put the phytol on and then monitor it everyday until the ph returns to normal salt water levels.

-Vlance

You are welcome, VLance! I hope we can work together again.

Please let us know how your Daphnia experiment turns out. You are just going to test phytol, right? No oil. The hydrocarbons in crude oil would likely affect Daphnia adversely in some way, but since phytol is part of chlorophyll and Daphnia eat algae which contains lots of chlorophyll, i would predict that phytol would not hurt them--at least in reasonable amounts.

Are there any other chemicals in your phytol? Phytol is a liquid that is insoluble in water but is soluble in organic solvents. I am wondering if the company you bought the phytol from sent it dissolved it in a solvent. Check the label of your phytol and make sure it is JUST phytol and not dissolved in some other chemical.

I am a little confused about your experiment to determine how long it takes for phytol to biodegrade. For one thing, if you just put phytol in distilled water you will not be measuring biological but chemical effects. There are many organisms in sea water that might be able to digest phytol but none in distilled water. Can you explain how a pH change shows degradation of phytol?

I'm asking these questions because they are questions that a judge or someone reading your report may ask you and you need to show them that you are aware of these issues and can explain how you justify the experiments you did.

Let me know if you have questions.

Sybee