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GC testing & the Purity of Essential Oils, Dr. Kevin Dunn, Professor of Chemistry
Derek asked me to talk about GC Testing and the Purity of Essential Oils and I said really? Are they really going to sit through GC testing and, yeah, yeah, that’s what he wanted so that’s what I’m going to talk about? This is a project that we started in the middle of last year. Derek wanted to test the essential oils from Essential Depot so he donated a chunk of money to Hampden-Sydney College which helped us to buy a new gas chromatograph and now I’m going to explain what we do with it. So first I have to say what is an essential oil? Probably most people here know what an essential oil is but there are lots of fragrances that aren’t essential oils so I want to be clear about what we’re talking about. First of all it’s volatile. What does it mean that it’s volatile? Many people think volatile means explosive or dangerous but to a chemist the word has a very specific meaning. All it means is it easily evaporates. It comes from Volare , to fly in Latin. All it means is that it’s easily evaporated, and if you think about it how you could smell something that isn’t easily evaporated, it would never get to your nose and so maybe you would psychically have a psychic smell but if you want a real smell in your nose it has to be volatile. It’s derived from some part of a plant. There are lots of fragrances that aren’t so we are talking about specifically something that comes from a plant. It carries the odor or flavor of the plant and it’s usually a complex mixture of aroma chemicals. We will talk about specifically some of the usual players. So now I need to talk about purity and again chemists always use words very precisely. In common language the word pure is talked about all the time. So you talk about, how about milk? Pure milk, what is pure milk? It’s milk that doesn’t have some non-milk things in it. That’s what ordinary people mean by pure but a chemist has to make it more difficult than that. A chemist would never talk about pure milk because milk is already a mixture of different things so we reserve that word for things that are only one thing. So there are compounds and there are mixtures and you’re familiar with some things that are compounds. Compounds have formulas, they have chemical names. So water is a compound, salt is a compound, sugar is a compound. So water has a formula H2o and a sugar that you may be familiar with fructose is also a compound has a formula C6H1206. Those are all pure substances. When I mix sugar and water together I get a strange thing called honey, and you might say “Oh this is pure honey,” and what you mean is it doesn’t have cane sugar added to it or something; but a chemist would never talk about pure honey because honey is already a mixture of fructose and water. Instead I would use for a mixture like that I would use the word it’s genuine honey, it’s authentic honey, it’s unadulterated honey, I would use all those words to describe honey but I wouldn’t talk about purity. All right, there’s the word, a terrible poly-Slavic word and I want you to at least appear to be intelligent and the first step is even being able to pronounce a word like this. So this is chromatography, chrom-a-to-graphy, chromatography. Let’s practice it, chromatography, chromatography, and so you know if you’re going to talk to people about chromatography you ought not to stumble over the word, so you might go to a dark corner somewhere and practice and if you don’t, but look if you say GC, I don’t know GC this and the GC that and somebody says chromatography and you say chromotoplagraphy. It just marks you as a charlatan, you know you’ve been throwing them out GC like you know what you’re talking about but you can’t even say the word, so practice that. It’s a technique for separating mixtures into their components. So for example you might do chromatography on honey to separate it into the sugar and the water portion. So we are going to do a demo now on all the project tables. You have a Mason jar with some water in it and you have a paper towel. This is a high-tech demonstration. You have to order this on line, it’s very expensive, but I just used paper towels that I happened to have in my kitchen. At the bottom is a pencil line and there are four dots here. These dots are from Flare felt tipped pens, so I have a purple dot, and a pink dot, and a green dot and a mystery dot, and I wrote the names of these in pencil because I’m about to put it in water and I don’t want my information to run, so I wrote that in pencil. So to get this started, you need to take your paper towel and kind of roll it into a little tube so that it will fit down in the Mason jar. We don’t want the water line to be above the pencil mark. We want the water line to be down here so that the water wicks up through the paper. You’re not just dipping it in, you want it to stand up so that just the very bottom of the paper gets wet. Question: Do we take it out? Kevin: Just leave it in there, it is going to take probably a half an hour to do its thing and we will visit it from time to time that you can see some of you that the ink is already starting to run but it is running in a very interesting way. Anybody need another one I got one more. Okay, so I really can’t get away from the nerds and cheerleaders. Everybody here has probably heard the nerds and the cheerleaders but it has kind of become an obligatory thing. So everywhere in the universe there are nerds and cheerleaders at high school cafeteria tables. They are always separated in the nerd table and the cheerleader table. Somebody who doesn’t know might think that nerds hate cheerleaders and that isn’t true because I knew cheerleaders and they liked me just fine, individually. And you might think nerds hate cheerleaders but that’s not true because I loved cheerleaders. So that’s not that the nerds and the cheerleaders don’t like each other it’s that the cheerleaders prefer the company of other cheerleaders to the company of nerds. You might think the nerds also prefer the company of other nerds to cheerleaders, it’s not true. It’s simply that when a nerd goes to sit down, if he sits down at the cheerleader table every cheerleader at the table can’t help thinking it could have been another cheerleader sitting there. So where the nerds sit wherever there are empty seats and so the nerds tend to congregate the cheerleaders separately, but that’s not what I’m talking about, that’s just kind of preamble, because now I want to talk about the cheerleader wedding reception and at the wedding reception there is a receiving line with the bride and the groom and their parents and all the bridesmaids and everything and in order to get to the food you have to run the gauntlet of this receiving line. So let’s imagine now the cheerleader wedding reception and the couple of nerds that got invited by accident, I don’t know. Somehow the nerds wound up at the cheerleader wedding reception. Who’s going to get to the food first, the nerds or the cheerleaders? Why is that? Because at the receiving line every cheerleader is going “Oh where did you get your nails done, oh your hair is so beautiful.” It takes 20 minutes to get from one person to the next if a cheerleader is talking to another cheerleader, but if a nerd comes, nerd says “Hey, what’s up?” The thing is to make this allegory work it’s necessary that you imagine that in the etiquette of this receiving line it is okay to cut. So the cheerleaders are all lined up they’re gabbing about all kind of cheerleader things and the nerds comes up and says “Hey, yah yah” the nerd gets quickly through the receiving line, has already finished eating before any of the cheerleaders have gotten through the line. All that’s necessary is the cheerleaders have greater affinity for other cheerleaders then they do nerds and so when it’s a cheerleader receiving line the nerds move quickly and the cheerleaders move more slowly. You could have the opposite situation, the nerd wedding reception with a couple of cheerleaders that have gotten invited and the same thing would happen because the nerds are all talking “Hey did you see that chess game the other day?” They are talking about all kinds of nerds stuff and the cheerleaders like “Yah, hey what?” and so the cheerleaders get through very quickly and the nerds are kind of working their way through the receiving line. So that allegory is going to serve us well when we talk about the “C” word. Audience: Chromatography. Dunn: Chromatography, very good. There are 500 guests, if there are 250 nerds and 250 cheerleaders, the nerds will get through first. So, we have the same thing going on here, but it’s not nerds and cheerleaders, we’ve got water and paper and some of these inks have a greater affinity for the water, some of the inks have a greater affinity for the paper, and so the different colors of ink move at different rates as the water moves up through the papers. We’re going to call the paper the “stationary phase,” so it stays still and we are going to call the water the “mobile phase.” Now these pens are all water soluble inks, they’re Flair felt tipped pens, but I could do the same thing with permanent markers but I would need to choose a different “stationary” and “mobile” phase. I wouldn’t use water as the mobile phase because how about permanent marker in water? Never gonna move. I would have to use a different solvent like alcohol in order to get a permanent marker to do the same thing that we are doing here. Compounds with greater affinity for the water move quickly. Things with the greater affinity for the paper move slowly. Now we’re going to add a word to it, gas chromatography. So now we’re talking not about water and paper, we’re talking about volatile things, we’re talking with things that are easily turned into the vapor. The stationary phase is a thin glass tube. It’s bigger than a human hair but it’s not much bigger than a human hair. It’s very a very thin long glass tube and it’s so thin that you can bend it. I meant to bring a piece but I forgot. You can bend it and it actually comes as a coil. It’s wound up on a little metal thing to hold it in place. The column that we’re using is 30 yards long and it’s about a 10th of a millimeter in diameter. It’s very very thin and very long. It’s not just a glass tube because the glass tube has to have something in it, the stationary phase, and there are lots of varieties for what the glass tube could be coated with on the inside. There are lots of choices and it depends on what is it that I’m analyzing and which stationary phase I’m going to choose. The mobile phase is helium gas. We have a steady supply of helium gas flowing through the tube and when the molecules are injected onto the column, they will move at different rates depending on their affinity for whatever stationary phase that I’ve chosen. This whole thing, the glass tube and everything is inside an oven and I can program the oven to start out at one temperature and to ramp at a certain rate until it reaches the final temperature. So the things that are not very volatile won’t move very quickly when the oven is cold but they will move more quickly when the oven is hot. Okay, this is what it looks like. We have the GC on the right and the mass spec on the left. I’m going to talk about mass spec later. I’m going to zoom in now. There’s the GC part of it. You can see the coil is the column. I have an injector, I have a syringe I use to inject a sample on to the column, and it moves slowly or quickly through the glass tube until it vents out the back end. Just like the receiving line at the wedding. This is RGC, it’s a GC mass spec, I’ll talk about the mass spec later, but you’ve seen this probably if you ever watch NCIS. It’s not just that Abbey has a GC mass spec. It has a name, major mass spec and not only that we have the same one, it is the same model that Abbey uses on NCIS. It’s very noticeable because it has that circle there on the left. When I saw it I said, “Ha, that’s the one we have.” So on the right is the GC and on the left is the mass spec and I will talk about those two parts separately. Okay, this is what a gas, here is another word for you “chromatogram.” It’s easier than chromatography because it has fewer syllables. This is a gas chromatogram and all it is like a visual display of that receiving line. So the things that come out early, like 6.5 minutes there, the things that come out early are the nerds at the cheerleader reception and the things that come out late are the cheerleaders at the cheerleader reception. It took them longer to get through the column into the mass spec which serves the purpose of our banquet at the wedding reception. Okay, old school do you see, we didn’t have mass spec, mass spec is something that we didn’t have when I was growing up, so all we have was GC, so all we really knew was how long does it take for this compound to move through the column. So you get a bunch of pure chemicals, pure using pure in the way that a chemist uses the word “pure”, inject them onto the columns, squirt them one by one onto the GC. You measure how long does it take to get through this glass tube, that’s called the retention time and then you make a table. This compound takes this long, that compound takes that long and any time you see a peak at 6.543 minutes, you know, “Oh you know, that compound that takes 6.543 minutes,” and you come up with a list of all the compounds that you’re interested in and how long they take to run the gauntlet. So this is an example of just a GC report, Beta myrcene takes 6.543 minutes, D-limonene takes 7.219 minutes and caryophylene takes 13.056 minutes. Just by measuring the retention time I can identify the compound. But there is a problem with the old school method, what if your mixture contains a new compound that you’ve never seen before. How are you going to identify that? How can you even begin in the infinitely many chemicals that there are, how you can even know which one I should search for. The second thing is what if two compounds, two different compounds have the same retention time. They’re going to come off right on top of each other, how are you going to know which one is which? So we are going back to the GC. We are going to move from the GC part to the MS part. This is a block diagram for the mass spectrometer and the idea is we are going to squirt whatever it is that’s coming off the column into a vacuum. We are going to bombard it with an electron beam. You can imagine shooting a machine gun at it and that’s going to fragment the molecule into parts and then the mass spec part, the rods there are going to determine the mass of each of the fragments. This crazy crazy way to identify a molecule but you know what you can’t just look at a molecule and see what it looks like, you have to be able to measure something about it and the mass is something that can be measured. So I’m going to talk about fragmentation patterns. If you were shown a demolished vehicle, would you be able to identify it? So you got a pile of car parts there. Can you tell the difference between a Toyota Corolla and a Tacoma? It’s just a pile of car parts. You might not know, what is a Corolla? It’s a small sedan. What is a Tacoma? It’s a little pickup truck. It is not a giant pickup truck. They are sort of the same size and so you are looking at a pile of car parts there, imagine though that you notice in amongst the pile of car parts was a tail gate. Can you identify it now? Audience: Yeah Dunn: Yeah that’s probably okay. I can probably say okay that’s a tail gate it’s got to be a pickup truck because the Corolla doesn’t have a tail gate. Now you might also look and there’s the Toyota logo and look here it says Corolla on it, but the molecule doesn’t have that. All it has is the weight, so now imagine you aren’t able to see a tail gate, you are only to see here’s a part that weighs this much, here’s a piece of the car that weighs that much and here’s a piece of the car that much and if you looked at the Corolla and a Tacoma, you would find things that weigh different amounts. If you are looking at the steering wheel, how about the weight of the Corolla steering wheel compared to the weight of the Tacoma steering wheel? Probably about the same and you know what you never find half a steering wheel. When you demolish a car, basically the steering wheel comes off all as a part. You never find half of a tail gate, the tail gate came off as the tail gate. The door, the windshield, all these parts came apart intact. Can you tell it from a Highlander? What is a Highlander? Answer: A guy with a kilt. Bigger. Dunn Yeah, so a Highlander is much bigger than a Corolla. You add up all the parts and my God, you can’t tell a Highlander from a Corolla, you’re not trying very hard because physically the pile is just bigger, the parts weigh more and there are more weighed parts when you add them up. How about from a Camry? Camry and a Corolla. Kind of similar cars. Camry an infinitely superior car. That’s what I drive. I looked at the Corolla, it just wasn’t up to snuff, Camry and Corolla, and Camry is a little bit bigger car. Its door, the Camry door weighs more than the Corolla door and how about a Civic? Okay, look these are almost the same car. The Toyota Corolla and the Honda Civic are Honda and Toyota’s attempt to reach the same market. The doors’ gonna weigh kind of similar. The windshields, the steering wheels kind of similar between those two cars. If you just had a pile of car parts and their weights, could you tell a Corolla from a Civic? You know if you had demolished both cars and weighed all the parts you might be able to tell the difference but you would have to scrutinize it a little more carefully. All right now we’re talking about molecules. We are going to demolish a molecule, what are the parts? So this molecule is Ethylbenzene. I don’t care that you know the structure of the molecule but it’s a ring with stuff hanging off of it. So the whole molecule is on the right there and it’s a six membered ring with CH2, CH3 hanging off of it, two carbon atoms. If you add up the weight of that molecule, it adds up to 106, but if you knock the CH3 off of it, that’s like removing the steering wheel, it only weighs 91. You’re going to find a mass peak at 91 and 106. You aren’t going to find one in between because that would be like removing half a carbon atom which just doesn’t happen when you are demolishing a molecule. There is a more complicated molecule. I don’t know if I can even pronounce this 3-pyr, blub, blub, bulb, I don’t even care if you can pronounce it. It’s a big honking molecule and when you fragment it, it fragments into the things that fall off easily and that means you break a bond so here I have the whole molecule all together is 369 mass units. But if I chop off that last little piece it only weighs 298 and if I chop off a couple more, it’s 272 and you can identify in green the mass fragments that you would expect to see for a molecule like this. Okay, so now we have the NIST Library, which NIST is the National Institute of Standards and Technology. They have a mass spectral library that a computer can search so when you see a fragment pattern it can look and see what molecules have fragment patterns similar to that. It is automatically searchable so we love this library - 242,466 compounds in the library which is really really nice. We have problems with mass spec, we always have problems. What if two compounds have similar fragmentation patterns, that would be like the Corolla and the Civic, very similar fragmentation patterns, and again what if the compound that you are trying to find isn’t among the 242,000 compounds in the data base? Okay so this is a mass spectrum of Alpha-Pinene. I don’t even care that you know what Alpha-Pinene is but when you smash it apart it falls apart into these fragments and I’m going to show you a similar fragmentation pattern for Beta-Pinene. There’s Beta-Pinene. I’m going to use two hands now, Alpha-Pinene, Beta-Pinene, Alpha-Pinene, Beta Pinene. You see they’re very similar but they are not identical. What do you see is the difference? Alpha-Pinene, Beta-Pinene. They have the same fragments but they have different numbers of those fragments. So Beta-Pinene, look at the peak at 42. Short in Alpha-Pinene, tall in Beta-Pinene. So if I were really really careful, I might be able to tell the difference between Alpha-Pinene and Beta-Pinene, but they are so similar, it’s kind of hard for the computer to look at these and say “Ah, the thing that you are interested is actually Alpha-Pinene”; but Alpha and Beta Pinene have different retention times. Alpha-Pinene comes out first, Beta-Pinene comes out second so while their mass spector are very similar, their retention times are quite different – 5.66 minutes compared to 6.93 minutes. So if I have a doubt now I’m looking at a mass spectrum, I look at the retention time and I’m trying to match is what I’m looking at does it have the right retention time and at the same time does it also have the right mass spectrum and those two together will be a pretty bullet proof identification for a compound. I also have compounds that have the same retention time. Okay in the old school do you see it is very hard to see the difference between D-Limonene, Beta-Phellandrene and Eucalyptol because they come out at like 7.2 minutes? They are right on top of each other, but their mass spector are very different. D-Limonene, the tallest peak is 68, Beta-Phellandrene is 93 and Eucalyptol is 43. So, even though their retention times are very similar, their mass spector are very different. Combining the two GC and Mass Spec gets me identification for a compound that I can rely on. Okay, when you go to the Essential Depot website now, when you look up an essential oil, you will be able to look at the GC report for not just in general lavender oil, but a batch specific. This batch of lavender oil that we are currently selling right now has this GC and you can tell how much of each of the components is in it. So this one has .45%, though the one we are looking at is the area percent and of course that’s very small so here it is blown up, 6.54 minutes, we’ve got .45% Beta-Myrcene, .7% Limonene, 4.63% Eucalyptol. If you think about lavender oil, what is it mostly? Mostly Linalool and Linalyl Acetate. Pretty much all lavender oils will have those two and the other things that they have depends on what country is it from, what season was it grown in and how was it distilled, all the various things that go into a lavender oil. The thing I want to show you though is that this is multiple compounds, it is not pure lavender oil because it is already a mixture and the composition varies from batch to batch. What I’ve listed here I think are 16 compounds, how many compounds are there in lavender oil? The answer is it depends on how hard you look, because the mass spec will find, if you want to find out to a 1/100th of a percent all the compounds that are there, even a 100th of a percent, the GCMS will do that. What we have decided mostly is to settle on 15 compounds for identifying the authenticity of essential oil, but a lot of places they do a 100 compounds and then they are left with a giant list of all the possible things that are in it. We are thinking this is a little bit more understandable if you are just trying to get a snapshot of this particular batch of oil. Question: So ideally, the fewer compounds there are the better the lavender oil is? Dunn: No, you couldn’t be farther from the truth. You also can’t say the more compounds there are because you know what, all I had to do is click a button I could have found 100 compounds instead of 15. So how do you know whether this is a good lavender oil or a bad lavender oil, you have to come back tomorrow because that is what I’m going to be talking about in tomorrow’s lecture is. Now that we know about GCMS and what the report means, how do we look at a report and decide, can’t say pure lavender oil, what would we be looking for? An authentic lavender oil, a genuine lavender oil, an unadulterated lavender oil, how do we know whether this is a good lavender oil or a bad lavender oil? Question: The percentages are they constant in the batch? Dunn: For example, if I took 8 different bottles from the same batch, would I get the same percentages. There is going to be a little bit slop, one of them might be .45%, one might be .46%. Would you say that’s a problem? Probably not. If I got .45% from one bottle of the same batch and 27.2% from the other, something’s wrong, that doesn’t add up, and even if I took the same sample and injected it 12 times, would it always come out .45%? No it is probably going to vary a little bit around, there is going to be a little bit of slop from measurement to another. Question: Isn’t it like wine tasting where you have this one expert and that expert and one likes the wine and the other thinks the wine is terrible. Dunn: There is a little bit of that but what I’m going to talk about tomorrow will help us to decide what we mean by an authentic Lavender oil. It’s a great question. Hold on to it for 24 hours and let it mature like a fine wine. Question: Are the results similar from one batch to another batch and from another batch? Dunn: No, they could be quite different and so for example, I could have an authentic Lavender oil and the next batch that comes in is adulterated. Well you wouldn’t expect them to be the same and we would like to be able to tell them apart but even if I had two different samples of a genuine lavender oil, they may be quite different and you will be surprised at how different they can be. I will be talking about that tomorrow and you will see. It is not enough to have one, I need multiple so we can compare. For lavender oil, for example, I compare Essential Depot lavender to 42 different lavender oils, because there are variation. Forty-two genuine lavender oils that are different from one another, now how do I tell this lavender oil that I’m looking at right now fits among the 42 other lavender oils. Again I will be talking about that tomorrow. Question: Are you talking about 42 different types of lavender? Dunn: Lavender from Bulgaria, lavender from France, lavender from the United States. Lavender from Bulgaria in the winter time compared to the summer time, compared to northern Bulgaria, southern Bulgaria. Audience Even from the time of day on the same plant. Dunn Again it could even that this is the same plant grown in the same field sent to two different distillers and they’re slightly different because the distillation process was a little different. So all those things come into play and make the job of deciding of whether it is authentic or not a much trickier question that you might think of at first blush. Question So if you have a batch of soap that you put lavender oil in you shouldn’t list it as pure lavender oil? Dunn: So everybody knows what pure means. If you use the word pure, everybody knows what you mean, but a chemist has a specific meaning to that word and so personally I don’t talk about pure lavender oil but I understand that you know people outside of chemistry they were using the word before chemists were, so I don’t really have the right to tell them that meaning of pure is wrong, it is just not the word that I would choose if I were trying to be precise. On a label you usually list the botanical name of the oil that you are using. Question: On the 1 oz. bottle we have 1oz lavender oil today. Ten years from now as long as it stays sealed would that have the same exact properties or is it like wine it could change? Dunn: It can change over time and how much does it change depends on was it stored in a cool dark place, was it in the attic. It can also be different for I would expect wintergreen. Wintergreen you can bury it in a time capsule and 100 years from now you come back and the wintergreen is still wintergreen, but it may change from oil to oil how stable it is over time. Question: Okay does the color of the bottle really matter? Dunn: Some of these will be degraded by sunlight. They typically come in a brown bottle to protect them from light. They are not black bottles because you would like to see how much is in there and so a brown bottle is a good compromise. A dark green bottle and you find the same thing with wine, right? You don’t generally find wine in a clear glass bottle because they are trying to protect it from sunlight. Or in the box is a perfect protection from sunlight. Question: Are there infinite number of variables that could affect the calculation any one time, any one situation? Dunn: I wouldn’t say infinite, but there are more variables then a lay person might imagine. So typically I don’t want to step on tomorrow’s lecture but there is an organization called ISSO. There whole job is to specify things, that’s what they do. So if you want a blender, what is a blender and they have a specification for and not just essential oils but all kinds of things. What is authentic peanut butter, okay? That’s what they do for a living is to make up rules for what genuine peanut butter is and typically ISSO specs contain a dozen different compounds they look at that it ought to be in this range and not in that range. Question: Okay, plastic or glass, does it make a difference? Dunn: Okay, it does and so I’m going to again let that question age like a fine wine and we will talk about it tomorrow. Question: Okay, looking at that chart I see what is Eucalyptol and camphor doing in Lavender? Dunn: Okay, when you die and get in heaven, you can ask God that question, “What, what in the world is a lavender plant thinking about when it makes Eucalyptol?” If you remember where do you think the word Eucalyptol came from? Eucalyptus, because somebody had to discover that compound first and they got to name it and they named it after the place where they found it in this case and it is also more terrible then that because Eucalyptol is generally known in the trade as 18 Cineole or any of another dozen synonyms. So now that multiplies my headache enormously because I see “Oh this should have Eucalyptol between 1% and 8% and I have 1-8% cineole.” They are the same thing and you have to learn that sometimes these compounds have multiple genuine names. So the question was “Why does the lavender plant produce Eucalyptol at all, I don’t know, ask the lavender plant.” And different lavender plants and, some lavender plants make it and some don’t and I don’t know why. I can’t really look in the mind of a lavender plant. All I know is that if you look at the 42 different genuine samples of lavender oil, some of them have Eucalyptol and some of them don’t and they vary in composition. So the data base that we use is academic journal articles about essential oils distilled in the laboratory from that plant right there that was grown in this field at that time of year and harvested and distilled. The answer is that it could be there from contamination but it is not necessarily there from contamination. Nothing is stopping me from having extra Eucalyptol to my lavender oil, but it doesn’t have to be the case. Question: Don’t they sometimes come in the same family, like you may not think about lavender being in the Eucalyptus family and then that’s probably why it contains some of it? Dunn: Yeah, but look at what else is in here. Limonene, where do you think the guy that discovered Limonene, where do you think he discovered it from? Answer: From lemons. Dunn: From lemons and limes, that is where the name comes from and yet you would expect lavender oil has some Limonene. GCMS is a powerful tool, blub, blub, blub. Tomorrow we are going to learn what the percentages mean and what they tell us about the authenticity of essential oil. Before I finish, go and pull your paper out and what do we conclude from this chromatogram and has developed over the last 40 minutes or so. Just pull it right out and we had four dots there, one was the purple dot, and one was the pink dot, the green dot and the mystery dot. Can you tell anything about the mystery dot? That is not the sort of the same pink, it is the same pink, but what else does it have. It also has the green because the mystery dot and then I wrote a green dot right on top of it. So we have a mixture of things. The purpose of the chromatogram is to separate a mixture into its components. Look at the purple dot. I didn’t mix the purple dot. I took the purple dot straight out of the pen and yet you notice something different about the purple dot. That purple dot, the ink in the purple pen is actually a mixture of at least two different inks because look we got two different colors there. Which one has the greater affinity for the water? Audience Purple Dunn: Blue or purple, greater affinity for the water. The blue had the great affinity for the water, the purple had the great affinity for the paper. They moved at different rates, so what looked like one ink is separated into at least two. How many compounds are actually there? Audience: Two Dunn: Notice I say at least two because do I know there aren’t three? It depends on how hard you look, just like the gas chromatograph, it depends on how hard you look, how many different colors you are going to find. I thank you for your attention and we will pick up tomorrow morning with interpreting GCMS reports.