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C60 concentrations from major vendors

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#1 kmoody

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Posted 19 January 2016 - 06:52 PM


Hello LongeCity community! We received around 30 samples from you to test in our laboratory, some home-brews and some from 3rd party vendors. We have completed HPLC testing for C60 concentrations within the samples. Several of you requested that your samples be made anonymous, so we have decided to release a compiled graph of concentrations measured from 3rd party vendors. To receive your individual sample results, please PM me and I will provide those to you. :)

 

Disclaimer: Ichor has conducted this testing under research grade conditions as a free service to the LongeCity community. Members of the Ichor staff hold equity positions in BioSenex, Ltd. None of these staff members were involved in the testing or handling of any of these samples. Neither Ichor nor any of its employees hold equity positions or receive compensation from any of the distributors listed.

 

Carbon60OliveOil.com

Concentration claimed: 800ug/mL

Concentration measured: 825ug/mL +/- 25ug/mL

Samples tested: 5

 

Vaughter Wellness

Concentration claimed: 900ug/mL

Concentration measured: 531 +/- 24ug/mL

Samples tested: 3

 

SES Research

Concentration claimed: 800ug/mL

Concentration measured: 563ug/mL +/- 19ug/mL

Samples tested: 9

 

c60concentration.png

 

We are still debating what, if any, additional analytical testing should be done. If you have input on interesting things for us to look for please post here and we will take your suggestions into consideration.


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#2 Turnbuckle

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Posted 20 January 2016 - 11:27 AM

Ironic that the company claiming the highest concentration actually has the lowest, and the company with the most technology has failed to correctly measure its concentration. One thus has to wonder if the purity of the dry fullerene materials are as claimed.


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Click HERE to rent this advertising spot for C60 HEALTH to support Longecity (this will replace the google ad above).

#3 DeadMeat

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Posted 23 January 2016 - 10:36 PM

Thanks very much for doing this.

Maybe UV/VIS(or just VIS) spectroscopy could be useful. To look at possible differences in adduct formation. And maybe it could be interesting to heat a sample. To try and simulate aging of the solutions and to see if something changes in the visible spectrum or with HPLC.

From Cataldo's book chapter 13.3.6:

The new absorption band at 435 nm in the C60 spectrum has been attributed to the 1,2 addition to the fullerene cage to the fatty acid chains either across to the double bonds by a Diels-Alder addition or, more simply, by radical addition (Cataldo and Braun, 2007). Thus, fatty acid esters are able to not only dissolve C60, but also react with this molecule causing the addition of the fatty chain to the fullerene cage. In fact, the bands at 435 nm shown in Fig. 13.3 appear only when C60 is stirred at 75°C for a couple of hours in the esters of fatty acids. Only for olive oil the new band appears much weaker than in the other cases and displaced at 450 nm (Fig. 13.3B). Since this oil contains chlorophyll, the displacement may be probably due also to a charge–transfer interaction between C60 and chlorophyll or with other impurities.

On standing in air, at room temperature the C60 fullerene solutions in vegetable oils are not stable, but change their colour from violet to reddish. The electronic absorption spectra show a gradual increase in the absorption band in the visible initially in the range between 450 and 550 nm. Similar results are obtained both by heating the solutions in air or under nitrogen. In the latter case prolonged heating is needed to achieve the same results. Heating C60 solutions in linseed or other oils for 15 minutes at 150°C (in air) causes the entire spectrum of C60 in the visible to disappear completely as shown in Fig. 13.3F.

 

Cataldo's 2010 study has some more info on color changes from violet to red to brown. And the types of adducts and possible epoxide formation that can be seen in the visible spectrum, when exposed to air.

 

Attached Files


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#4 sensei

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Posted 23 January 2016 - 11:37 PM



Ironic that the company claiming the highest concentration actually has the lowest, and the company with the most technology has failed to correctly measure its concentration. One thus has to wonder if the purity of the dry fullerene materials are as claimed.

 

 

Were the samples taken from manufacturer sealed UNOPENED containers?

 

The only way to be certain that samples are undiluted (for whatever reason) is to use samples taken in the Lab by Ichor personnel from unopened manufacturer sealed containers.

 

Just Sayin'


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#5 Mind

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Posted 24 January 2016 - 06:40 PM

You will have to trust me on my sample. I agitated my manufacturer's container first (to make sure it was mixed well), then put a sample in a different container, so I think it was representative.


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#6 sensei

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Posted 24 January 2016 - 09:41 PM

You will have to trust me on my sample. I agitated my manufacturer's container first (to make sure it was mixed well), then put a sample in a different container, so I think it was representative.

 

 

I'm not distrusting anything or anyone.

 

Merely pointing out that the normal protocol is to evaluate samples taken directly from unopened manufacturer sealed containers.

 

VW can easily argue adulteration if that is not the case.  And based on the VW website VW is very litigious.

 

Ichor may want to post a disclosure that the samples were provided by individuals and they cannot ensure no adulteration occurred before they received the samples.


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#7 ambivalent

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Posted 25 January 2016 - 09:16 AM

We can be pretty confident, though, given the variance is low - all the samples would need to have been adulterated in an almost identical fashion from independent sources.


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#8 Turnbuckle

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Posted 25 January 2016 - 12:21 PM

 

You will have to trust me on my sample. I agitated my manufacturer's container first (to make sure it was mixed well), then put a sample in a different container, so I think it was representative.

 

VW is very litigious.

 

 

 

 

Are you sure? Have they actually sued anyone about anything?


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#9 sensei

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Posted 26 January 2016 - 05:49 AM

Serious question:

 

Using HPLC to determine concentration requires a minimum of 4 calibration sires.

 

Peak area and height of the calibration samples at the selected wavelength(s) is used to create a calibration curve.

 

Olive Oil is documented to have widely varying constituents by volume WRT fatty acids.

 

We know different fatty acid adducts of C60 emit (reflect) light at varying wavelengths.

 

Some of us on this forum have seen how a light filtered olive oil will result in a pink-violet-magenta oil, but a darker extra virgin oil results in a dark merlot/cabernet/berry brown spectrum.

 

Is it possible that the 2 manufacturers identified as having lower concentrations of C60   ACTUALLY simply emit less in the calibration solution peak wavelength(s) because of a difference in olive oil fatty acid makeup?

 

Differing wavelength emission spectra for C60 dissolved in oil is identified for olive oil that has a higher chlorophyll concentration (darker greenish extra virgin olive oils), as well as C60 in oil that has been heated to a certain temp.


Edited by sensei, 26 January 2016 - 05:58 AM.

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#10 niner

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Posted 26 January 2016 - 05:14 PM

This is an important thread and we don't want to see it cluttered with off-topic posts.  I just deleted one, and will be moderating this thread a lot more heavily than usual in order to keep it on track.


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#11 kmoody

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Posted 26 January 2016 - 07:42 PM

Thanks very much for doing this.

Maybe UV/VIS(or just VIS) spectroscopy could be useful. To look at possible differences in adduct formation. And maybe it could be interesting to heat a sample. To try and simulate aging of the solutions and to see if something changes in the visible spectrum or with HPLC.

 

Cataldo's 2010 study has some more info on color changes from violet to red to brown. And the types of adducts and possible epoxide formation that can be seen in the visible spectrum, when exposed to air.

We think along the same lines. :)

 

We have done all of these experiments already (completed a few weeks ago). UV/VIS simply isn't sensitive enough to see the differences. We did not note any changes in our home-made C60oo after 

 

 



Ironic that the company claiming the highest concentration actually has the lowest, and the company with the most technology has failed to correctly measure its concentration. One thus has to wonder if the purity of the dry fullerene materials are as claimed.

 

 

Were the samples taken from manufacturer sealed UNOPENED containers?

 

The only way to be certain that samples are undiluted (for whatever reason) is to use samples taken in the Lab by Ichor personnel from unopened manufacturer sealed containers.

I agree 100%. This data is not intended to say anything about what was received by the vendors. It is merely intended to report, in aggregated form, what we received from the community. I have no way of verifying even if the vendors listed were actually where the person submitting the sample received their C60oo from. So as a stand alone I would not extract too much information from these observations.

 

Serious question:

 

Using HPLC to determine concentration requires a minimum of 4 calibration sires.

 

Peak area and height of the calibration samples at the selected wavelength(s) is used to create a calibration curve.

 

Olive Oil is documented to have widely varying constituents by volume WRT fatty acids.

 

We know different fatty acid adducts of C60 emit (reflect) light at varying wavelengths.

 

Some of us on this forum have seen how a light filtered olive oil will result in a pink-violet-magenta oil, but a darker extra virgin oil results in a dark merlot/cabernet/berry brown spectrum.

 

Is it possible that the 2 manufacturers identified as having lower concentrations of C60   ACTUALLY simply emit less in the calibration solution peak wavelength(s) because of a difference in olive oil fatty acid makeup?

 

Differing wavelength emission spectra for C60 dissolved in oil is identified for olive oil that has a higher chlorophyll concentration (darker greenish extra virgin olive oils), as well as C60 in oil that has been heated to a certain temp.

This is a fantastic question. You are completely correct for UV/VIS spec analysis. However, for HPLC we do not run the C60oo directly. We extract the C60 using toluene and run that with the mobile phase, so olive oil composition does not matter here unless it changed the C60 so much that it elutes immediately or is retained for longer than the run time. Either is possible but unlikely.


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#12 password

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Posted 27 January 2016 - 02:50 AM

Are the SES research samples mostly homemade?  As far as I can tell from reading these forums, most people avoid the pre-made SES C60oo because of the potential negative effects of sonification.  The most common protocol for homemade C60oo seems to be 1g/1.5L, or 660ug/ml for 99% purity C60.  If that's what is being measured, an average of 563ug/ml is kind of low (85% of expected), which really puts into question the quality of SES dry fullerene.

 

Kelsey, did you also measure the concentration of C60 in the C60oo that you bought premade from SES?


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#13 Pyrion

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Posted 27 January 2016 - 10:27 AM

I find it encouraging that all vendors at least *do* have C60 in it. Although i am a bit disappointed about VW's performance, i will continue to buy it, mostly because i live in europe.

 

This is speculating, but i think one reason why concentrations are lower than adverstised may be that because of too high demand, they might have shortened the mixing process... so maybe they had the advertised concentration earlier.

 


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#14 DeadMeat

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Posted 27 January 2016 - 02:03 PM

This is a fantastic question. You are completely correct for UV/VIS spec analysis. However, for HPLC we do not run the C60oo directly. We extract the C60 using toluene and run that with the mobile phase, so olive oil composition does not matter here unless it changed the C60 so much that it elutes immediately or is retained for longer than the run time. Either is possible but unlikely.

 

Since toluene is non polar and aromatic. If you add enough toluene, I can understand that you would get most of the pristine C60 out that way. But wouldn't something as a triglyceride C60 adduct just stay in the olive oil?

Did you already try to just dissolve/dilute everything, in DMSO or maybe the toluene + acetonitrile used as mobile phase and HPLC that? Or would that mess up the column?



#15 niner

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Posted 28 January 2016 - 02:34 AM

Since toluene is non polar and aromatic. If you add enough toluene, I can understand that you would get most of the pristine C60 out that way. But wouldn't something as a triglyceride C60 adduct just stay in the olive oil?

 

The solubility of pristine c60 in triglycerides is quite low, so the vast majority of it should be reacted with the unsaturated fatty acids.  I would expect that a triglyceride would have reasonable solubility in toluene, but the presence of a fullerene on it would be a significant force pulling it into the toluene.  Since the same protocol would have been used with all samples, seeing the expected amount of c60 in the carbon60oliveoil.com sample suggests that the methodology is correct, or at the very least that the VW sample has significantly less c60 than the carbon60oliveoil.com sample.



#16 sensei

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Posted 28 January 2016 - 02:46 AM

This is a fantastic question. You are completely correct for UV/VIS spec analysis. However, for HPLC we do not run the C60oo directly. We extract the C60 using toluene and run that with the mobile phase, so olive oil composition does not matter here unless it changed the C60 so much that it elutes immediately or is retained for longer than the run time. Either is possible but unlikely.

 

 

 

It would be interesting to compare the UV/VIS to the HPLC.

 

disclaimer -- I am not stating your process is faulty or wrong in any way.  I work in a field that requires evidence for verification, validation etc.  

 

I'm not completely convinced that toluene will [consistently] strip all the C60  or fatty acid adducts out of [every] sample of olive oil.

 

I have no insight into the ability of toluene to cleave C60 from a fatty acid once an adduct has been formed, or separate out the C60 adducts as well.

 

I have seen no data presented that the adsorbents utilized are consistent [re: deposition] with respect to all fatty acid adducts of C60.

 

I have seen no data regarding the possibility that there are varying degrees of pristine C60 and C60 fatty acid adducts that are deposited on the adsorbent.

 

I have seen no data regarding the chromatographic profile of pristine C60 vs C60 plus fatty acid adducts of C60.

 

The potential uncertainty regarding exactly how the analytes [C60 vs. C60 adducts] are deposited/adsorbed by the adsorbent coupled with the potential uncertainty regarding the chromatographic signatures of C60 and C60 fatty acid adducts, raises a serious and significant question in my mind regarding the accuracy of the reported concentrations.

 

Honestly, this is NEW SCIENCE, and kmoody ( Kelsey?) and his friends at Ichor are on the cutting edge so to speak.

 

Please don't take this post as me beating up  kmoody or Ichor.  I am raising serious scientific questions regarding how to quantify the concentration of C60 in olive oil via chromatography ( UV/VIS or HPLC)


Edited by sensei, 28 January 2016 - 03:15 AM.


#17 DeadMeat

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Posted 28 January 2016 - 02:12 PM

 

Since toluene is non polar and aromatic. If you add enough toluene, I can understand that you would get most of the pristine C60 out that way. But wouldn't something as a triglyceride C60 adduct just stay in the olive oil?

 

The solubility of pristine c60 in triglycerides is quite low, so the vast majority of it should be reacted with the unsaturated fatty acids.  I would expect that a triglyceride would have reasonable solubility in toluene, but the presence of a fullerene on it would be a significant force pulling it into the toluene.  Since the same protocol would have been used with all samples, seeing the expected amount of c60 in the carbon60oliveoil.com sample suggests that the methodology is correct, or at the very least that the VW sample has significantly less c60 than the carbon60oliveoil.com sample.

 

 

If almost all of the C60 in olive oil was in the adduct form and most of it was extracted with the toluene. Wouldn't there have been a difference in retention time with HPLC? Compared to the pristine C60 in toluene, that I assume kmoody used as a reference?

I could be misinterpreting this, but from Cataldo's book chapter 13.4. At least in mole fraction, he considers the solubility of C60 in vegetable oils to be quite high. He also claims it is in agreement with his theoretical calculations. And I don't think Cataldo considers adduct formation with unsaturated fatty acids to be the driving force for dissolution at room temperature. He describes it more as something that just happens to some extend, while the C60 is already dissolved.

 

@kmoody:
Are there any triglycerides or other olive oil components detectable with HPLC in the toluene after the extraction?



#18 niner

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Posted 29 January 2016 - 01:53 AM

If almost all of the C60 in olive oil was in the adduct form and most of it was extracted with the toluene. Wouldn't there have been a difference in retention time with HPLC? Compared to the pristine C60 in toluene, that I assume kmoody used as a reference?


I could be misinterpreting this, but from Cataldo's book chapter 13.4. At least in mole fraction, he considers the solubility of C60 in vegetable oils to be quite high. He also claims it is in agreement with his theoretical calculations. And I don't think Cataldo considers adduct formation with unsaturated fatty acids to be the driving force for dissolution at room temperature. He describes it more as something that just happens to some extend, while the C60 is already dissolved.

 

Yes, I'd expect a difference in retention times between a triglyceride c60 adduct in toluene and pristine c60 in toluene.  Kelsey, is that what you see?  I'd also expect to see some triglyceride that had no c60 eluting at yet another time.

 

Cataldo and others' takes on c60 solubility in vegetable oils is puzzling.  Those are unsaturated oils, and we know that c60 reacts with alkenes.  Cataldo and others don't seem to have considered that the reaction occurs at room temperature, just more slowly than if it's heated.   We need an unsaturated c18 triglyceride-- glycerol stearate, in order to see what the actual solubility looks like.  Here is a paper that looks at c60 solubility in a large number of solvents.  They don't have any triglycerides, but they have n-decane, which holds .071mg/ml.  Adding 8 more carbons will increase solubility, and the polar triester backbone will probably decrease it.  Having two more chains will probably increase solubility.  That's pretty vague, but I'd imagine that glycerol stearate would take up at least a few tenths of a mg/ml, maybe more.  Where it gets tricky is that at the same time, there are those reactive centers on all the unsaturated fatty acids.  I did a rough calculation using density of olive oil = 0.9, MW of olive oil = 896, and an average of 3 double bonds per triglyceride molecule, and 1 c60 per double bond as a way of calculating the maximum amount of c60 that could react with 1 ml of olive oil.  I got 2 grams, which is over a thousand times the amount that we see in practice.  That means that the unsaturations are available in substantial excess, so I think that they are going to suck up almost all the c60 that manages to dissolve.   It's certainly possible that something else is going on, so we should let the experimental results be our guide, whatever they may be.



#19 DeadMeat

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Posted 30 January 2016 - 04:35 PM

Yes, I'd expect a difference in retention times between a triglyceride c60 adduct in toluene and pristine c60 in toluene.  Kelsey, is that what you see?  I'd also expect to see some triglyceride that had no c60 eluting at yet another time.

 

Looking back on the Baati study, I think they already compared it with pristine C60 in toluene. It's not really clear what they mean by "extracted spectra". But from ref. 30, I assume it's UV/VIS, MS or both.

The resulting C60-olive oil solution is purple and contains 0.80 +- 0.02 mg/ml (n = 6) as determined by HPLC [30] after appropriate dilution in the mobile phase. The chromatographic profile and the extracted spectra of these solutions are similar to those obtained with a control C60-toluene equimolar solution. The stability of both oily and control solutions stored at ambient temperature and in the dark was checked monthly during 48 months. No change was recorded under our chromatographic conditions.


By the way, that ref. 30 (Moussa 1997) study also used toluene extractions before HPLC. So at least Kelsey using that method makes perfect sense. :)

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#20 4ever_young

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Posted 08 February 2016 - 01:46 PM

Thank you so much for the professional approach you have taken with these tests.

 

Your efforts are worthy of our respect, and the observations and comments of the members are valid and noteworthy.

 

It seems that the formulation process may indeed be corrupted by high demand from sales resulting in a somewhat shorter time frame than is needed for total dissolution of the C60.

 

It seems that the color does indeed tell the whole story.

 

 



#21 Turnbuckle

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Posted 08 February 2016 - 04:08 PM

 

 

It seems that the color does indeed tell the whole story.

 

 

How did you come to that conclusion? According to the paper in post #3 above, the color changes gradually from violet to brown--

 

The stock solutions of C60 in the various fats were stored in closed Durham glass flasks having a volume of 100 ml each, in presence of air. On standing a gradual change in colour was observed in the C60 solutions from initial violet colour to reddish to orange-brown to brown. At this final stage the colour of the solutions appears stable.

 

 



#22 BieraK

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Posted 09 February 2016 - 06:21 AM

Nobody sended a sample from buckylabs?



#23 Wilberforce

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Posted 09 February 2016 - 05:53 PM

The last two bottles from VW I've thrown away the last 0.5cm because it appeared cloudy, dark and viscous.  I'm now going to shake the bottle before each use, but my question is would this be consistent with C60 coming out of solution?  If so, why now?



#24 4ever_young

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Posted 09 February 2016 - 06:13 PM

Once the C60 is fully dissolved in the Olive Oil, it should not come out of solution. If not fully dissolved the C60 will settle to the bottom.

 

Freezing the solution will change the appearance, but once at room temp it should return to normal color and consistency.

 

The only change you should be seeing in a C60 that is pure and fully dissolved, is the OXIDATION of the C60 that DEGRADES the effective anti-oxidant properties of the C60.

 

As stated earlier in this forum this occurs over time and causes the color to turn brown.

 

Fresh C60OO at room temp is cherry red with lavender highlights, with smaller quantities being more Lavender.


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#25 LucidMind

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Posted 10 February 2016 - 07:10 AM

Does room light oxidize C60 or only UV light?

 

Is the oxidation of C60 from opening the bottle briefly to use the dropper eventually cause the C60 to become toxic? So if it starts to brown, then it's time to toss it?

 

 



#26 mikeinnaples

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Posted 11 February 2016 - 08:22 PM

Once the C60 is fully dissolved in the Olive Oil, it should not come out of solution. If not fully dissolved the C60 will settle to the bottom.

 

Freezing the solution will change the appearance, but once at room temp it should return to normal color and consistency.

 

The only change you should be seeing in a C60 that is pure and fully dissolved, is the OXIDATION of the C60 that DEGRADES the effective anti-oxidant properties of the C60.

 

As stated earlier in this forum this occurs over time and causes the color to turn brown.

 

Fresh C60OO at room temp is cherry red with lavender highlights, with smaller quantities being more Lavender.

 

Not sure I agree "completely" about the color as that can be pretty dependent on the chlorophyll content of the oil.


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#27 DeadMeat

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Posted 13 February 2016 - 11:18 PM

As for adducts being extractable with just toluene. In Moussa's 1998 study, that found retinol C60 adducts in the liver. They used a acetone + toluene mix for extraction. Instead of just toluene like in their 1997 study, that could not detect any retinol adducts in the liver. I'm not sure if this was the only major difference though, since the retention time of pristine C60 seems more than twice as long in the 1998 study.
https://www.research..._in_mouse_liver
 

Hepatic extracts obtained from C60-treated mice contain several additional compounds less hydrophobic than C60, which elute before C60 when analysed by means of reversed phase high performance liquid chromatography (HPLC) [Fig.1 (A)]. These compounds do not form in hepatic extracts of control mice when C60 is added to liver homogenates, indicating that they are not formed during the extraction procedure[Fig. 1(B)].

 

Then, the resulting homogenate was mixed with 2 ml of acetone and 1 ml of toluene and the mixture agitated for 30 s at room temperature in the dark. After centrifugation at 3000 g and -4 °C for 10 min, the supernatant was collected and evaporated at 38 °C under a stream of nitrogen. Finally, the dry residue was dissolved in 0.2 ml of the mobile phase for hepatic extracts of treated mice or 1.0 ml for control samples, before injection of 0.1 ml into the chromatograph.

For retinol and retinyl ester analyses we used the same mixture of acetone-toluene; 15 min of agitation allowed lipid fractions to be extracted. The dry residue obtained after evaporation of the supernatant was then dissolved in 0.5 ml of methanol before injection of 0.1 ml into the chromatograph.

 

Chromatographic analyses of C60 and its derivatives were performed at ambient temperature as described previously:8 isocratic elution (acetonitrile-toluene, 58 : 42, v : v) at 0.8 ml min-1.

Analyses of retinol and retinyl esters in hepatic extracts were achieved using a gradient elution (100% methanol during 10 min, then from 0 to 30% toluene in methanol within 10 min and 30% toluene in methanol during 10 min) at ambient temperature and 0.8 ml min-1.


#28 Junk Master

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Posted 16 February 2016 - 03:01 AM

For what it's worth, I felt a much stronger effect from c60oliveoil.com versus Vaughter Wellness.  Plus, I much preferred the olive oil used by c60oliveoil.com.


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#29 aribadabar

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Posted 16 February 2016 - 03:10 AM

For what it's worth, I felt a much stronger effect from c60oliveoil.com versus Vaughter Wellness.  Plus, I much preferred the olive oil used by c60oliveoil.com.

 

Did you mean carbon60oliveoil.com because if you go to c60oliveoil.com it gets re-directed to buckylabs.com?



#30 MissMaggie

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Posted 28 February 2016 - 10:31 PM

I'm curious if any information about the homebrew concentrations is going to be made public? I'm interested to know how much higher or lower concentration they might be compared to the commercial vendors. I'm assuming there's probably a wide variance?
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