What does peak area % mean?
@Deleted User @verticity @Gourmet Hawaiian Kava @Kalm with Kava @Palmetto @Rick.Sanchez
A strange table (comparison of what gets extracted by different solvents)
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What does peak area % mean?
@Deleted User @verticity @Gourmet Hawaiian Kava @Kalm with Kava @Palmetto @Rick.Sanchez
Hi Henry, peak area % is a representation of separate substances within a mixture on a printed chart, called a chromatogram that is produced by chromatography. The size of the peak area is nearly equal to the quantity of the substance present in the mixture.
Aloha.
Chris
Aloha.
Chris
thanks Chris. But does it mean that water actually extracts more compounds than some of the organic solvents? very strange
Yes it does. Each extraction process yields different amounts of different substances, It is interesting to see the differences in each method.
Aloha.
Chris
verticity
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The paper that figure comes from uses GC-MS (Gas Chromatography - Mass Spectrometry).
In this technique, a sample is vaporized in helium gas and passes through a narrow capillary tube made out of some kind of silicon polymer. Depending on the chemical and physical properties of the different molecules, such as how polar they are, they tend to stick to the column more or less. The different molecules take a different amount of time to pass through the column, so they are separated in time into "clumps". The "retention time" in that table is the time it takes for the clump to pass through the GC column. After they go through the column the molecules are ionized by electron impact (or some other method), then the ions go through a mass spectrometer which separates the ions according to their mass. One way this can be done is by passing the ions over a very strong magnetic field so that their path is bent different amounts depending on their molecular weight. Another way, which I think they used in this paper, is to pass the ions through a "quadrupole" which is 4 metal rods with an oscillating electric field, which basically lets you dial in a particular mass you want to look at. So the peak area is just the total amount of stuff that passes through both the chromatography column and the mass spectrometer. It is similar to HPLC, except that in HPLC, the sample is in a liquid instead of gas, and the detector is a UV light instead of a mass spectrometer. They can identify what each molecule is by comparing the retention times and masses with libraries of data that they have.
OK, anyway, so to answer the actual question: why do the peak area numbers for kavalactones appear to be higher for water? Because those peak areas are percentages, not absolute values. It is kind of misleading to list them side by side like that. Water extracts fewer chemicals than acetone. For example, if you look at all the kavalactones that are detected, in the water extract there are 9 kavalactones detected, whereas in the acetone extract there are 15 kavalactones detected, so in the latter case, each individual KL is a smaller percentage of the total stuff detected. In fact, acetone actually extracts about 3 times more kavalactones than water, and is also more efficient at extracting each of the individual kavalactones, as you can see in this table from the same paper:
I am a little suspicious of these results, though, because the ethanol numbers look way too low. Ethanol should extract more than water...I'm not sure what the deal with that is.
In this technique, a sample is vaporized in helium gas and passes through a narrow capillary tube made out of some kind of silicon polymer. Depending on the chemical and physical properties of the different molecules, such as how polar they are, they tend to stick to the column more or less. The different molecules take a different amount of time to pass through the column, so they are separated in time into "clumps". The "retention time" in that table is the time it takes for the clump to pass through the GC column. After they go through the column the molecules are ionized by electron impact (or some other method), then the ions go through a mass spectrometer which separates the ions according to their mass. One way this can be done is by passing the ions over a very strong magnetic field so that their path is bent different amounts depending on their molecular weight. Another way, which I think they used in this paper, is to pass the ions through a "quadrupole" which is 4 metal rods with an oscillating electric field, which basically lets you dial in a particular mass you want to look at. So the peak area is just the total amount of stuff that passes through both the chromatography column and the mass spectrometer. It is similar to HPLC, except that in HPLC, the sample is in a liquid instead of gas, and the detector is a UV light instead of a mass spectrometer. They can identify what each molecule is by comparing the retention times and masses with libraries of data that they have.
OK, anyway, so to answer the actual question: why do the peak area numbers for kavalactones appear to be higher for water? Because those peak areas are percentages, not absolute values. It is kind of misleading to list them side by side like that. Water extracts fewer chemicals than acetone. For example, if you look at all the kavalactones that are detected, in the water extract there are 9 kavalactones detected, whereas in the acetone extract there are 15 kavalactones detected, so in the latter case, each individual KL is a smaller percentage of the total stuff detected. In fact, acetone actually extracts about 3 times more kavalactones than water, and is also more efficient at extracting each of the individual kavalactones, as you can see in this table from the same paper:
I am a little suspicious of these results, though, because the ethanol numbers look way too low. Ethanol should extract more than water...I'm not sure what the deal with that is.
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True. But doesn't it still mean that water extracts more than ethanol under certain conditions?
worrier
Kava Curious
Would this mean that boiling your kava (possibly in bulk) would result in a more potent solution?
verticity
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Yes.
verticity
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I actually don't see anything about heating in the description of how the extracts are prepared. GC-MS does involve heating, but that happens after the extract is prepared (full text attached).
So basically (cold?) water extraction is actually not just qualitatively, but even quantitatively better than using ethanol? It's just a matter of using the right kind of equipment?
verticity
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As far as I can tell that is what this article claims. I find it extremely hard to believe. It is peculiar that they did not detect any flavokavains in their ethanol extract (and didn't detect any FKC or FKA in their acetone extract). This suggests there was some problem with their extraction methodology because it is possible to extract all the FKs with ethanol, as described in papers like this one:
Oliver Meissner and Hanns Haberlein, J. Chromatography B, 826(2005) 46-49
I might be wrong, but I think Dr Schmidt argues that ethanolic extracts are safe (in comparison to the acetonic ones) at least partially because they contain much less fkb. But I cannot find any quotes in his papers so either this was in one of our private conversations or I made it up lol.
verticity
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Even in that paper it looks like acetone is more efficient than boiling water (and even a bit more efficient than pressurized super heated water)
verticity
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That might be true, but the paper I cited above shows that FKs are easily detectable in ethanolic extract at least (it doesn't compare with other solvents)
(Paper attached - See Figure 2B)
verticity
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Correction: in the above paper it does say something about ethanol possibly being more efficient at extracting FKs than dichloromethane (which is a solvent similar to chloroform...basically chloroform for wimps, since it is not as bad of a carcinogen as chloroform is...)
Palmetto
Thank God!
@verticity the numbers listed in the table do not appear to be percentages. Look at the last two rows on the bottom, where the sums don't add up to what you would expect would they were percentages.
And I used to love dichloromethane as a solvent for lipids. I once cleared out a building when moving large bottles of chemicals, and 10 L of chloroform spilled on the floor. That's when I ditched chloroform.
And I used to love dichloromethane as a solvent for lipids. I once cleared out a building when moving large bottles of chemicals, and 10 L of chloroform spilled on the floor. That's when I ditched chloroform.
verticity
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The paper states that the numbers are "the percentage of peak area". It is not really clear about what they are percentages of, though. I had assumed that they did something like the following:
1) For each retention time, integrate over the entire mass spectrum to plot a chromatogram of "total stuff" versus retention time.
2) For each peak in the chromatogram, measure it's area, and normalize to the total area of all the peaks in the chromatogram.
3) Look at the mass spectrum corresponding to each retention time to identify the molecular weights.
But if this is what was done, then why don't the percentages add up to 100 (except for acetone, which is pretty close to 100)?
I don't really understand why. It is possible the percentages would not add up if there were peaks in the chromatogram that could not be identified, or if the chromatogram has a baseline that is not subtracted out. Or maybe they normalized everything to the HPLC analysis for kavalactones that they also did, instead of to the GC chromatogram itself. The paper is not very clear about exactly how they calculated those numbers. Maybe it's a standard GC thing that I don't know about because I am not an expert in that technique, I don't know.
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verticity
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Oops. You mean you had everyone who has passed out from the chloroform carried out on stretchers...?