I suggest that you spend some time trying to decipher the mass spectra of the compounds you are trying to identify. What novel features are in the mass spectra? Are there losses you can identify? Is there a suspected molecular ion-- if so, try searching something like Chemfinder.com for that molecular weight. Based on the identification of other peaks in your extract, are there similarities in the mass spectra that might put you on the trail to an identification? Some molecular drawing packages will calculate ion masses, so draw some likely structures and try to match up with ions in your experiment. Spend some time with the data and see what you can figure out without a library.

Comparative tlc using standards

My guess is if the NMR is not a match, it is not the compound that you are expecting. The NIST database is a great tool but not perfect. It is only and indicator, not a certainty.

With NMR 1H, 13C, 2D... IR, MS, you should be able to find the structure. Comparative TLC, HPLC is ok if you can get a reference compound. good luck

How pure is this fraction? If your fraction is reasonably pure, mainly one compound, then NMR is the ultimate instrument. Have you tried HPLC-PDA-MS/MS? The UV spectrum and the MS fragments will provide excellent information on your fraction.

Well is it possible that the components your are trying to identify are not in the NIST library. In that case, you might want to try commercial vendor MS Search program/software

NMR either proton or carbon 12 and also infrared spec which will give the type of functional group present.

Calculate retention indexes and compare them to those of the same column type as the one that you are using. This procedure often eliminates many uncertainties. Also, try and follow the exact chromatographic method that was used for the determination of original retention indexes in the library.

You can get certain information from your sample performing certain qualitative test to differenciate between families of compounds. Simple experiements like solubility and classification tests will help you in identifiying functional groups. That way you'll have some structural information of your sample before choose the best separation-detection system for your sample.

You can get certain information from your sample performing certain qualitative test to differenciate between families of compounds. Simple experiements like solubility and classification tests will help you in identifiying functional groups. That way you'll have some structural information of your sample before choose the best separation-detection system for your sample.

First of all you should run HPLC so that you can get an idea whether the fraction has one or more compounds (indicated by peaks with different retention times). If it is more than one compound then I suggest going for purification (silica gel or solid phase extraction), check the individual fraction and the go for mass spectra (ESI-MS) of the fraction with activity. Since you are not getting the compound identification in GC (as the library has limitation). Now you should go for NMR (1H, C13 , HMQC, HMBC, COSY and NOSY). The result of these analyses will surely give you information about your unknown compound. Based on mass spectra you can also look at the literature and find whether there is some compound with the same mass and shows the same activity.

First of all you should run TLC to know that whether the fraction is having one or more compounds.Then its melting point, FTIR , UV , GC/MS, MNR etc

I appreciate you all for your contributions. Shubhankar, I am going to try the demo version. In my online search for answer to my questions I came across the acllabs. But I didn't pursue it further because I couldn't buy the software. But I will see how far I can go with the demo version. Thanks.

Certainly, the demo version has limitation. Some laboratories now provide online structure elucidation to help other researchers.

Yes Daryono, I agree with you. Can you provide me more information on how I can link up with any of those laboratories?

Did you try by using H/D solution exchange? If your molecule has acidic hydrogen atom ( such as an alcholic group...) you can obtain information on the CAD fragmentation pattern

You may try to caontak Prof. Oliver Fiehn with link: http://fiehnlab.ucdavis.edu/

My concern, you must make sure that the isolated compound should have high purity.

maybe I would suggest coupled or tandem analytical techniques using other non routine detectors, presumable you should be looking at an oil or related compound

I will suggest you to use HPTLC facility to characterize the compound present in plant fraction. This instrument is very much useful for medicinal plants.

I agree with the others that determinng purity is important. Another powerful software tool is is Mass Frontier Version 7.0 which is provided by Thermo. However, this software is expensive

Maybe your fraction is a mixture of isomers, library search will give you some suggested structures anyway. You could try differect columns before other experiments.

or try isometric separation techniqe such as ion mobility spectrometry

PLANT FRACTION usually are mixtures consisting of monoterpene and sesquiterpene hydrocarbons, their oxygenated derivatives, and aliphatic oxygenated compounds.

The difficulties that arise with these in the GC-MS peak identification of these complex samples is due to the fact that many terpenes have identical mass spectra. This is a consequence of similarities both in the initial molecule, or in the

fragmentation patterns and rearrangements after ionization. Hence, MS

identification of these compounds should always be accompanied by mass spectral identification that may support the MS library search results.

And sensitivity of Mass is far higher than IR and NMR

What is the the value of match answer in NIST library? It must be 900 or higher. If it is the case, you have isomer of the NIST suggested compound. Formaceck did some essential oils analysis with NMR (13C), but of course the best is to get a pure compound for NMR analysis. For essential oils analysis, GC-FTIR could solve the problem. Unfortunately, Agilent doesn't produce anymore this instrument. Some labs still have it.

With the structural information from NMR data, you can try to do the fragmentation based on m/z.

NMR is one of the best ways to go

If you want to screen many compounds, you can also run LC-MS (ESI or APCI) to get molecular ion and some structural information (MS/MS). There also some databases you can access to compare that information

I thank everybody who has contributed so far. But I wish to make the situation clearer. I am aware of many other options that I can adopt to get good results, apart from the GC-MS, as I stated when I posted my question. The issue here is that I have already run GC-MS (with NIST library) of my plant fraction . Unfortunately, some of the peaks do not correspond to any of the compounds in NIST library. I have recently been trying to use AIST library. I still intend to use other methods to analyse the fraction. But, for now, I want to know other resources or databases that are available to help me identify those other compounds that are not present in the NIST lib. Guillermo Labadie, could you let me know those databases that I can access to compare the information. My main interest and desire is to know those databases, preferably those that are free. Thank you.

Unfortunately for ESI-MS there are not good databases like NIST, but you can have good information like Chemspider,

Sorry to say but I think that you are stock in a NO through road. You need to start looking outside the box, I mean.... can you separate/isolate some of these peaks using a different techniques. The answer is yes, and you will have to. HPLC might the next move, if available.

I heard about preparative GC, not sure what it is worth?

Good luck

I suggest that you spend some time trying to decipher the mass spectra of the compounds you are trying to identify. What novel features are in the mass spectra? Are there losses you can identify? Is there a suspected molecular ion-- if so, try searching something like Chemfinder.com for that molecular weight. Based on the identification of other peaks in your extract, are there similarities in the mass spectra that might put you on the trail to an identification? Some molecular drawing packages will calculate ion masses, so draw some likely structures and try to match up with ions in your experiment. Spend some time with the data and see what you can figure out without a library.

Thank you so much Elin Ulrich. You have a perfect understanding of my problem. The advise you have given is just the way out. Already, I have started trying to come up with some structures based on the MS fragmentation patterns, while cross-checking with MS spectra of compounds of same molecular weight using AIST library. I will try Chemfinder.com. I hope the package is free. Later on, I intend to put the fraction through other chromatographic and identification procedures. What I am trying to do is to enrich my knowledge by trying to find various available means of solving the problem I highlighted in my question. I don't want to just set aside the gc-ms spectrum just because I cannot find some of the compounds in NIST library. I believe this is the way to learn. And, honestly, I have already learned a lot in the process of finding a solution to this challenge. Although, this method is very tedious and time consuming, it has greatly enriched my knowledge.

Dear Dr Eseyin,

I suggest to [1] you can enhance the search option using NIST; zoom the peak of interest, get the MS average at the top 1/3 peak hight, subtract from average of MS baseline, then search NIST. [2] Dried sample + vial + 20 uL MSTFA of BSTFA, close, heat at 80 C for about 15 min, cool, inject. Now you have O-TMS or N-TMS derivatives, which could easily identified by NIST.

You have (perhaps unfortunately) encountered the reality of compound identification by mass spectrometry (either GC-MS or LC/MS). If the spectra are not in a database, then they have to be interpreted manually, and this is not always easy! It requires skill and experience. And remember - even if there's a good match with a database spectrum - there will always be some doubt unless you have an authentic standard to directly compare. Even then you'd need additional information, e.g., NMR, which may even be impossible, since you need more sample for NMR than you need for mass spec. Always keep a seed of doubt in your identifications unless you have a lot of supporting data.

I haven't used this myself so don't know the overlap with NIST, but Scripps have a database called Metlin (http://metlin.scripps.edu/)

from the website I quote

"The METLIN Metabolite Database is a repository of over 75,000 endogenous and exogenous metabolites that includes metabolites from essentially any living creature, whether it is bacteria, plants, human/animal, etc.,... METLIN is also unique in that is has comprehensive tandem mass spectrometry data on over 11,000 molecules. The information is provided to facilitate metabolomics experiments and metabolite characterization. "

Maybe this could be of some use

Good luck

Chris

Chris Barber, thanks for your fantastic contribution. I have just checked the site now and I think it will be very useful.

Hi Chris & Olorunfemi, please take care to compare apples with apples here. Metlin (to my knowledge) contains ESI-MS/MS spectra so spectra from even-electron fragmentation, whereas GC-EI/MS (which I assume you are using) produces odd-electron MS/MS spectra from the Electron Impact Ionization which might not really compare. You want to make sure that you are looking at comparable experiments.

Thank you for the information, Chrstof Lenz.

NMR, HPLC-MS; those are the most common

Hi, Olorunfemi Eseyin, GC is used for the volatile and sem-volatile organic compounds, while NMR for almost all organic compounds, which can result in your different results based on the two techniques. Therefore, I suggest that you can use high resolution MS coupled with LC ( such as LC-QoF or LC-orbitrap ) to separate the plant fraction and indentify every peaks based on accuracte quasi-molecular and fragment ions. LC-NMR/MS is also a good choice.

For the identifications of volatile compounds on GC-MS, don't miss to use the traditionnal rules to calculate the Kovats' Index of the unknown molecule and to compare it with the tabulated indices on the same GC phase. Indeed on both phases should be more relevant. By this way, a collection of evidence could lead you to the final structure. And finally read the Mac lafferty's book will be fine.

Good luck.

I do not know what types of compounds you are looking at but a lot of information can be obtained using UV/Vis spectroscopy and shift reagents for many plant compounds. These are classic phytochemical techniques.

After really analysing your mass spectra and identifying possible fragmentations etc.

You could do GC-MS-MS to confirm these "parent-daughter" relationships, confirm the fragments and identify molecular weight etc.

Obviously this may mean making a number of GC-MS-MS runs but this is easier than isolating quantities for NMR.

If you are analysing complex natural mixtures there is a high probability that incorrect identifications, or failures to find a library match for a particular peak are due to incomplete resolution of different compounds by the chromatographic separation. This leads to the spectrum of the peak being a mixture of the spectra of two (or more) compounds. These mixed spectra are not in any library, and are impossible to interpret (although you may be able to recognise that the spectrum cannot have come from one compound).

Try running your samples on a column with a different stationary phase, this may separate components that were co-eluting in your original analysis. The most elegant way to do this is with two-dimensional GC, but that requires special instruments.

We have had luck with complex mixtures isolated from biological sources by doing MS-MS analysis. First, we tune it to a standard of the compound, which I am not sure if you have the capacity to do, but maybe you have a related compound. You can titrate how hard you are ionizing the compounds to maximize a parent peak - usually a higher MW peak to allow for greater secondary fragmentation. You can then ask the machine to isolate that MW peak and fragment it again, which should allow you side-by-side analysis of the complex mixture MS and the pure compound MS. Some compounds respond better to CI, rather than EI, on the first ionization, which allows for a softer ionization and thus greater higher mass peaks.

Once you have fine-tuned your spectras, you can easily screen larger groups of complex mixtures and pull out just the compound you are looking for. I've found this to be as useful as GCxGC techniques, but doesn't require as specialized of instrumentation and as large of sample size (maybe it's me, but GCxGC seems to require more sample to do the analysis).

Good luck!

You have more than enough information to work with by now . Just incase this has not been mentioned, "REMEMBER THE ADDUCTS'.

Cheers.

Kokoette,

Adducts in EI GC-MS ?? I don't think so.

Well, when you are trying to isolate or identify a phytochemical, always study the plant and you will have some broad idea, as to the nature of the substance you are looking for. Then correlate this with the application that you want to investigate. This will give you some range of chemicals that will be the nearest possible compound. Now finally try to see how the thermolysis have occurred and try to add the mol.wt to get the final compound. Ultimately look for functional groups that the compound might have, by means of IR. Also try get the absorption maxima from a reliable UV/VIS study. NMR analysis will be of very little help as you are working with a mixture of compounds. NMR works best when the compound is very pure.