Identifying Unknowns - The proper way - Discussion thread

Hash and Stuff Distillation
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I’ve seen several posts or individual threads requesting information on unknowns or asking what unknowns are in their reaction or even something as ridiculous as “Here is some black tar what is it?” I think a thread that specifically focuses on discussion of proper techniques and steps to take in order to perform the proper identification would be very useful. I will start out with a high level look at the most important (in my opinion) techniques used for proper unknown identification.

  1. Separation and Preparatory Chromatography - This is an essential tool in your arsenal for identification of unknown molecules. By utilizing proper chromatography you will be able to isolate every individual molecule in your mixture and collect those fractions. This should really be step one, especially if you are running a synthetic reaction and have your final product. Take your final product through liquid chromatography separation and collect each peak that elutes. If you have peaks that look like they coelute, collect the coelution as one fraction and rerun it under different parameters, with the goal to separate those. Identification of unknowns is much easier if you have the ability to provide isolated unknowns for the following steps.

  2. Proton and C13 NMR (both 2D and 3D NMR depending on your unknowns) - The workhorse in the unknown identification space is NMR. I believe benchtop NMR has become much more affordable or attainable but this is really the core technique to utilize. NMR will allow you to, molecule by molecule, determine your chemical structure. More complex molecules such as proteins will benefit heavily with 3D NMR but 2D NMR is adequate for small molecule analysis. If you have been able to confidently isolate your unknowns through step 1 above, you will have a much easier time leveraging NMR for an exact structure (both molecular formula and structurally where each atom goes).

  3. LC/MS and GC/MS - I believe that while NMR and Mass Spec are very complimentary to each other, it serves better as a support tool to NMR results vs a stand alone technique. If you are dealing with something completely unknown besides assumptions, you can compare your fragmentation patterns to various libraries…but often times libraries will provide several matches and in the realm of potential cannabinoids not being identified, they may not even be present as a library selection. The data gathered from mass spec combined with the structural data gathered through NMR will allow you to completely identify your unknown molecules.

There is definitely a lot of legwork that needs to get done for all of this to come together, and unless you have access to your own benchtop NMR, this often times is a limiting factor in the whole process and why most people (understandably so) try to default to identification of an unknown solely through mass spec.

If you truly want to figure out what that unknown 10% is in your D8 synthesis or your conversion reaction, looking into these three steps and techniques would be the place to start.

edit I’m happy to get into more detail on these techniques or provide good publications to start with, but I wanted to keep things rather short to start with as a reference to the ideal steps to get into. I wanted to avoid something that looks overwhelming and unapproachable to people who may not be so analytical chemistry inclined

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I dig it, i think there’d be a lot of value in what could be added to the thread.

May I politely suggest sort of front-loading this with the more simple methods of analysis that are available to those who DON"T have access to such methods (which is most people)? Ie, ‘Step 1: Attempt to dissolve a sample in water, ethanol, and heptane, note which dissolves and which does not’, etc. etc.

I realizing you’re shooting for ‘actually figuring out what these compounds are to the chemical/class of compounds’, and the lower-level methods don’t make these any less ‘unknowns’ but most people are looking for ‘how do i deal with/remediate this’.

If we feel like this is already addressed in other threads to satisfaction, feel free to take or leave the suggestion. just feel like it’d make for a more comprehensive discussion <3 looking forward to this one developing

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The problem with any analytical project is the complexity of the matrix.

You’re not getting anything from NMR until/unless you can isolate individual components.

The best I would recommend, as an analytical chemist, would be shotgun mass spectrometry. That is used in biochemistry to unravel complex matrices. You’d need a database of phytochemicals and some AI to query it. Again, out of reach for most people and finding a facility that could analyze a schedule I compound (e.g., not receiving any federal grants for any purpose) isn’t exactly easy.

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Isolating your individual components comes through proper preparatory chromatography, which I agree can be very complex…but that’s the nature of trying to properly identify a completely unknown product.

If you can achieve proper isolation and collection you should have only the following items now combined in said fraction:

  • Peak of interest collected from start to end of peak (the unknown)
  • Mobile phase (known matrix)
  • Diluent used in your sample preparation (known matrix)

Where there are concerns of coelution, you can run individual fractions back through under a different stationary phase, or even potentially different chromatographic conditions of temperature, flow rate, pH of mobile phase, etc… to further potentially separate and isolate coeluting peaks.

Everything here is a known except for item 1 which then allows you to begin the steps of NMR and Mass Spec analysis.

I completely agree that isolation of your component is extremely important and I also agree that the complexity of work that goes into how to go through this process the right way to end up being confident with your identification is much greater than I think most people consider.

I think the discussions surrounding this topic can help to really inform people how difficult it is to identify with accuracy and precision, the actual structure and identification of unknown products or byproducts, especially unknowns produced in synthetic reactions that may not have significant data published from either private sector or academic research.

The whole process is something that is, as you said, most likely not easily attainable by most people or organizations - which further supports that people performing these different synthetic reactions and creating a 90% pure product with 10% being unidentified with any certainty, and then putting it out into the market, creates a potential consumer safety situation that may or may not have acute or chronic implications.

I think one goal of this thread I’d like to have is for people performing these reactions to be sure to do their due diligence on the final purification step post reaction.

I’m sure the implementation of a final chromatography step could help bring that 90% purity product up to a 99% purity product for most synthesis reactions. Often times your major peak of interest can be isolated through preparatory/flash chromatography and then concentrated back down removing the mobile phase, leaving you with a high purity end product of your desired result with significantly less concerns about those unknowns produced as they now would not end up in the product.

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