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Torsten

Need help with GC/MS specs

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Help!!

I have never used a GC/MS and only have a bare understanding of what they do. (I do know that they are an excellent tool for working out what's in plants though wink.gif )

Anyway, looks like I have managed to get together enough investors to buy one for Wandjina by the end of the year. Have done quite a bit of research on different models, their applications and the technology used. But it is not enough.

This is a major investment and I am responsible for making the right decision. I am way out of my depth here and need some help. Seems like most scientists merely know how to operate one, but have no clue of the different technologies available and what their respective advantages and disadvantages are.

Basically we need a machine that will handle good separation of complex mixtures (eg total plant extracts, alkaloid extracts, complex essential oil mixtures). Trace detection will not be required in most cases, however the unit should not be too specialised for bulk components as occasionally small amounts may need to be separated and detected.

If anyone can help, please either post here or e-mail me.

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To me it is a black box, though not necessarily black or a box. Hope that helps. smile.gif

[This message has been edited by theobromos (edited 19 July 2002).]

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To me it is a grey box, or set of grey boxes. That's the problem wink.gif

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Ok, I have just finished a semester of being introduced to analytical techniques so i should be able to give some feedback... Firstly, GC basically separates chemicals on the basis of volatility. You inject a sample at the start of a long hollow capillary tube that may be coated on the inside with a very thin layer of some liquid that acts as the stationary phase. Once you've injected the sample, the machine gradually increases the temperature inside the tube while an inert carrier gas flows through the tube/column towards the detector (in this case, the mass spectrometer). As the temperature inside the tube reaches the boiling point of each chemical fraction, they boil off the stationary phase and merge with the carrier gas to be carried quickly to the detector (where hopefully you will gain enough information to identify what sort of chemical just came off). The problem i can see with using this method for the applications you mentioned are that:

Most GC machines only go up to about 200-400 degrees celsius, so if the boiling points of the chemicals you inject are higher than that, you can fuck the column. Say you wanted to analyse various salvias for salvinorin analogs... you couldn't use a GC because most of these big, relatively polar compounds only MELT at about 250 degrees. When working at such high temperatures, you have to also consider the thermal stability of the compounds of interest. GC would be the best choice for analysing essential oils and other low boiling point plant fractions. You could use it for quantitative analysis of the various aromatic ethers you might find in the lauraceae or monimiaceae for example. Or maybe you could separate the isomers of citral in lemongrass (like we did for a practical session).

Another problem with using MS any time is that it only offers very limited information for identifying new compounds and elucidating their structure. To really determine structure, you need 1H NMR and maybe 13C NMR, aswell as MS. MS would be useful for confirming the presence of compounds for which the mass spectrum has already been recorded. Your computer will record the MS as the compound comes off in the GC, and you can cross match this result with databases of MS for all kinds of chemicals on the internet. If you are looking for a particular chemical, you could easily obtain the known MS, and look for that MS in one of the fractions.

Another limitation of GC is that its only separating factor is volatility and you can't really change the nature of the stationary or mobile phases much.

Since you want to be able to have a high level of versatility and want to be able to look at alkaloids (probably with relatively high boiling points), i think you really need a HPLC / MS. With HPLC you can really control things and may separate chemicals by all sorts of chracteristics. You could analyse non-polar essential oils by using a normal phase column, and then the next day analyse alkaloids with a reverse phase column. You can control the polarity of the mobile phase during elution to analyse whole plant extracts. You can even add all sorts of weird coatings onto the stationary phase to do highly specialised separations. The same limitations of MS apply though. At most, you will determine the molecular weight and clues as to various functional groups present if you come across a new chemical.

I'm not sure what the price difference is between GC and HPLC machines devices, or all the nitty gritty when it comes down to accessories etc., but GC would be perfect for essential oil analysis... no good for alkaloids or other high boiling point chemicals. If you just want to focus on essential oils... go for GC.

If you really want to analyse alkaloids, you will need HPLC, and someone who really knows how to get the most out of one. Proper HPLC use is an artform that takes a lot of experience and knowledge to develop.

ideally, you would have both a GC and a HPLC since that would eliminate the hassles of changing your column each time you want to look at a different sort of chemical mixture. GC is simple and fast aswell as good for quantitative analysis. I think its also cheaper and would be a good start until you can make the most of HPLC. There is plenty of stuff to look at with gc, and it will keep you bisy for a long time.

I hope i haven't just told you a whole pile of information you already knew. there is a book at the library all about choosing the right hplc equpiment for you, if u want me to get a reference... alkaloids seem more interesting than essential oils. wink.gif

fractal

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OK, I see I'm going to have to go into more detail. (which is good)

The GC temp range of the model I am lookingn at is from 30 to 450 deg C. This easily covers most PEAs, IPAs, tryptamines, indoles and essential oils. Obviously the alkaloids need to be in freebase form. Am I missing something here??

The package comes with the NIST database covering 135,000 compounds. This is unlikely to cover everything, but upgrades are cheap and the database increases dramatically every couple of years. Failing all else I guess we can make our own reference spectra and reference concentrations by ordering pure compound samples from sigma etc.

I don't think HPLC is possible at this stage simply due to finances and the fact that the investors want a GC/MS. There are plenty of choices even in the GC/MS field to really fuck this up though, which is why i need more info.

My main concern has been the type of technology used in the MS. Most MS in use these days are of the transmission quadrupole type. The one I am looking at is a quadrupole ion trap machine though, which has some limitations. Quadrupole ion traps have a disadvantage in that they can hold only a finite number of ions; and when this number is exceeded, the resolution necessary to obtain a mass spectrum is lost. I understand this concept, however i don't understand what bearing this has on the actual operation. Does this mean the ion trap machine cannot handle samples with high ionising components? or does it mean that if one component is highly ionising, that it won't be able to read trace amounts with lesser ionisation?? Or am I completely on the wrong track here?

It appears that the ion trap technology is the most popular of the new machines bought these days and is replacing the transmission quadrupole technology.

The other technology that keeps cropping up is TOF (time of flight). This appears to be used for detecting trace amounts only, but some of the literature is a little confusing. It appears to give the best resolution, doesn't have the ion storage limit (that the ion trap has) and appears to be cheaper. However, is this machine even usable for 'general purpose' analysis??

I would be most appreciative if someone could descramble these different technologies for me please. There are a total of 5 technologies around, but I don't think the others are in the running anymore.

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If you plan to put crude plant extracts through a GC it will get really expensive, each new column costs about 500 USD and any simple plant extract made with alcohol, ether, or what have you will burn out a column in one use due to all the resins, gums, tars, chlorophyll, etc. So for each test you'll have to do extensive purification in order to spare your columns.

(The authorities might mistake this for illicit drug manufacture if they find out.)

Also, as you pointed out you'll need analytical standards. Getting all the permits for DMT, mescaline, etc. standards is very hard and very expensive. The standards themselves are also expensive.

I sure hope you know what your doing, I'd hate to see SAB go bankrupt.

Aren't there analytical chem labs in Australia that will do your analysis for you (for a not too obscene fee)?

That may be cheaper and FAR easier.

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Ok. I didn't realise that freebase alkaloids had such low boiling points. Sounds like you would have a better idea than me as to what you need...

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Originally posted by squiresk:

Getting a headache yet Torsten?

k.

yes! And I thought that wasn't supposed to start until AFTER I buy the bloody thing.

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Originally posted by Auxin:

If you plan to put crude plant extracts through a GC it will get really expensive, each new column costs about 500 USD and any simple plant extract made with alcohol, ether, or what have you will burn out a column in one use due to all the resins, gums, tars, chlorophyll, etc. So for each test you'll have to do extensive purification in order to spare your columns.

That's no problem (I mean purifying rather than buying new columns). Good to know though.

(The authorities might mistake this for illicit drug manufacture if they find out.)

Hmmm, maybe I am giving the wrong impression here, but I am actually more interested in the legal things and in plants that are completely unknown (no intent provable). I don't really want to find the highest alkaloid strain of acacia etc.

Also, as you pointed out you'll need analytical standards.

yes, but only if I want to do quantitative analysis.

Getting all the permits for DMT, mescaline, etc. standards is very hard and very expensive.

Had no intentions of working with these substances. I am more interested in things like the complex indoles.

The standards themselves are also expensive.

yep, just noticed that.

I sure hope you know what your doing, I'd hate to see SAB go bankrupt.

It's not going to be for SAB, but for Wandjina Gardens. The risk is shared by the investors. basically, even if I fuck this up, its still not the end of the world (or SAB). It would just make me look really stoopid wink.gif

Aren't there analytical chem labs in Australia that will do your analysis for you (for a not too obscene fee)?

You say this as if such services are available elsewhere. They are mostly not. Sure, the occasional lab will do such work, but at huge fees. I've been talking to many of the leading ethnobotanists around the world and there is a concensus that more than 95% of ALL ethnobotanical samples collected never get tested, even though most of them are handed to analytical labs. Of the ones that are analysed, they are usually old and often misidentified/mixed up by the lab. There are people in australia who have been waiting for results for several years. Even if the results came through now, they would be unreliable due to their age.

Christian Raetsch has given up collecting a lot of samples simply because none of them ever get analysed. Sasha would be sitting on a huge mountain of materials waiting to be tested (everybody sends him plant samples). The Wandjina GC/MS will be at least 50% dedicated to non-commercial ethnobotanical analyses.

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"Also, as you pointed out you'll need analytical standards.

yes, but only if I want to do quantitative analysis."- Torsten

Ooooohhhhh NO!

You got it backwards, you can easily determine the concentration of every component without A. standards (concentration is proportional to area of squigle. [area of single squigle divided by total area of all squigles times 100 = % concentration.])

You need the standards to IDENTIFY each component. These machines dont analize the structure of the components, they just give you a bunch of squiggly lines based on polarity, size, and concentration. The standards are used for comparison. If the squigle on a yohimbine standard matches one of the squigles on a extract, that shows that the extract has yohimbine. You cannot identify the structure of an unknown without doing a run (in identical solvent, temp, etc.) with a standard containing that compound. If its an unknown that you dont have a standard for you will never be able to determine its structure with GC (thats what IR spectrometers and various NMR scans are for, but the unknown must be isolated in pure form from the origional extract to do the tests, and youll never be able to buy a good NMR they are VERY VERY VERY expensive and dangerous [if you have a surgical pin in your back it can pull it out through your chest!!! Metal in the walls? Your lab IMPLODES!] IR specs on the other hand are very cool, and not TOO expensive.)

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Originally posted by Auxin:

Ooooohhhhh NO! You got it backwards

That's how it was explained to me by at least a couple of people - I think.

you can easily determine the concentration of every component without A. standards (concentration is proportional to area of squigle. [area of single squigle divided by total area of all squigles times 100 = % concentration.])

That is what I thought initially, but was then corrected. One of the replies was: "No because each substance has a different sensitivity. For a mass spec it depends on how ionizable the molecule is."

So brainiacs, which one is it?? Can we come to a concensus please??

You need the standards to IDENTIFY each component.

Isn't that what the database is for?? Sure, I will need a standard if the database doesn't have the matching data, but for at least 135,000 compounds I won't need it.

You cannot identify the structure of an unknown without doing a run (in identical solvent, temp, etc.) with a standard containing that compound.

so what is the database for??

If its an unknown that you dont have a standard for you will never be able to determine its structure with GC

In the first few years we will be concentrating on known compounds in new plants. Then later we might go looking for new compounds in old plants wink.gif

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my mind is dazed from all that I've read so far.... I do not even have a reference in my grey mass to comprehend some things discussed here.

But I am hungry for knowledge and hope to catch up!

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In the first few years we will be concentrating on known compounds in new plants. Then later we might go looking for new compounds in old plants wink.gif[/b]

And fish heads?

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is this in reference to the dreamfish???

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Resolution

For a detector to distinguish between ions differing by one unit eg NH3+ and CH4+ (m=16 and 17) the instruments resolution should be about 16. If we are looking at masses of 220 and 221, the resolution jumps to about 221.

Analysers

The original and most massive MS is the magnetic sector analyser. These are very good for high resolution analysis. For example C2H4+ and CH2N+ both have a mass of 28 but their exact mass is 28.0313 and 28.0187. Resolution required is 2200.

Quadrupole

Very compact, less expensive and more rugged, low scan times, by far the most common.

In a magnetic sector analyser ions are dispersed simultaneously like a diffraction grating. In contraast the quadrupole is analagous to variable narrow band filter. At any set of operating conditions, only ions with a small m/z (mass to charge) ratio are transmitted, the others are blocked.So it can be thought of as a mass filter rather than an analyser.

Most units can resolve ions differing by one unit upto over 3000 m/z ratio.

Ion Trap

The ion trap analyser is a device in which gaseous ions can be formed and confined by extrended periods.RF energy voltage is scanned and ions of specific m/z ratios destabilize and enter a detector.

They are cheaper than quadrupole and can resolve one mass unit for mass ranges of 500-1000.

They are apparently very good analyzers. With fourier transform, they are excellent instruments capable of a mass range of 12-2000 with a resolution of 50k-760k.

What to use them for

High res MS can give you exact MW down to a few decimal places.

Scructural information from the fragmentation (must have a lot of experience to be effective, and it is impossible for complex molecules).

Compound identification using a library.

Quantitative analysis. This requires a standard of the same compound being tested, or one which is very similar. Select one or more m/z peaks and the area under them will be proportional to their concentration. So for DMT and MeO-DMT, a standard of DMT may be used for both, if an appropriate peak is chosen.

If you need more information, call labs or universities and ask to speak to the mass spectrometrist. Im no expert and this is all I know.

[This message has been edited by Alchemist (edited 23 July 2002).]

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