Sinensetin has two protons which give two singlets (H3 and H8) on a 300 MHz. Using an higher frequency only H3 may reveal long range coupling with the two orto protons on ring B and give a triplet like multiplet.

To elucidate the structure I suggest to run also a 13 C-NMR.

Thank you very much, Dr. Venditti. Your response cleared my confusion. Your point directly makes sense in the actual 1H NMR spectra. Anyway, I already have a set of NMR determinations for the structure elucidation (1D: 1H, 13C & APT as well as 2D: HSQC & HMBC).

Just a follow-up, would there also be a major difference in the 13C spectra of sinensetin between the 600 MHz and 300 MHz NMR readings?

Muripaga,

Since you are working on natural products chemistry, you need the NMR as a strong tool to identify your compounds. I advise you reading

---A Complete Introduction to Modern NMR Spectroscopy by Roger S. Macomber, Wiely (1997).

Well, you will find out that at low frequency one expect second-order spin-spin system and as you go up to high frequency you could obtain a clean first-order spin-spin spectra. See the attached file just as an example.

Good luck

I truly appreciate the insight, Dr. Amer. Thanks. Now, I have one more reference to read on for me to help me fully elucidate the structure of the compound I isolated.

Dear Zakariya, as Alessandro Venditti wrote higher frequency NMR equipment will provide more aqurate and clear spliting specially for long range splittings than can help for defining neiburhood between hydrogens for geminal and vicinals although it may not be so different from lower frequency NMRs in C-NMR. however it really is valuable for researchers around natural products which are trying to elucidate structures.supposing your extracted compound probably is a known phytochemical, you had better take DEPT 90 & 135 in addition to H-NMR, C-NMR to compare with literature to make final decision.

Another point taken, Mr. Zarei. Thanks for the additional info.

Rule of thumb.......higher frequency better resolution in NMR.

Mr./Miss/Mrs. Muripaga,

with increasing of the working-frequency, you increase the resolution of the NMR instrument, i.e. capability to resolve closely disposed NMR signals. For natural product chemistry due to often complexity of 1H spectra such increasing of the resolution would be resonable, but let's do not forgot that the NMR is not absolute method for structural determination.

Mr. Amer,

Generally for natural product chemistry the set of robust methods is 1) chromatography; 2) mass spectrometry, which also provides structural information in gas- and condense phase; including solid-state; and 3) single crystal X-ray diffraction, including instruments for small molecular and proteing crystallography. Instruments in 3) ensure absolute structural determination in solid-state, including info for chiral configuration).

No doubt that NMR has its strong contribution to the structural analysis. But, when you have absolute structure and configuration by single crystal X-ray diffraction, than NMR even is not need. Of cource the chiral natural products crystallized very difficult and generally their bad crystal growth prevent utilization of single crystal X-ray diffraction. Same is valid for proteins. But for such molecules the methods of mass spectrometry also provide a highly accurate structural information.

As well as the cost of one single crystal X-ray diffraction instrument vary within 250 000 - 500 000 EURO (small molecules) and about 700 000 - 1 Mln Euro - for proteins. By contrast, the high resolution NMR instruments cost 700 000 - 4 Mln Euro. In addition the consumable costs of chromatographic, mass spectrometric and single crystal X-diffraction instriments are smaller than those for NMR ones. Therefore when you can be obtained a highly accurate (even absolute) structural information via mass spectrometry and single crystal X-ray diffraction, which encompasses gas- and condense phase (solution, solid-state, crystalline state), than utilization of above investments 600, even 900 or 1000 MHz NMR instruments for small molecular analysis, even for proteins has not logic.

In addition, the structural solving of proteins particularly by NMR is enough complex, even more complex, than this of solving the absolute crystallographic structure by protein single crystal X-ray diffractometer.

So, that for small molecular structural analysis the utilization of high resolution NMR instruments is extremelly expensive both as basic price of the struments as well as costs for consumables and support, like for the protein research, accounting for high complex for solving procedure.

Meaning high-to-very highe instrumentals, consumable and support costs as well as labour costs and time consuming for an essencially 'not absolute' structural method.

… well for X-ray crystallography you need a crystal. Without that you are out of luck. There are many instances where you cannot get a crystal (amounts, purity, heterogeneity, NON structured molecules). Sometimes you get the "wrong" structure i.e. Z DNA. e.g. B in solution, Z in crystal. Then there is protein dynamics !, domain orientation, Ligand binding (SAR) in solution. etc. Try do to this with X-ray. Yes there is a "logic" in doing high field NMR. FYI I am also doing X-ray crystallography.

For natural product chemistry: if you have enough PURE material by all means do X-ray. especially if you have access to the instrumentation. The advantage of NMR is that you do not need pure compounds and you can also run (LC-NMR). The disadvantage is that you need the expensive equipment and trained personnel. It would not make sense to buy all that to get a single structure. It also would not make sense to buy a MS or X-ray diffractometer for a single structure.

My point is that in either case you would most likely work with people/facilities that have the resources. Quite often you want a combination of techniques anyway. For example, some of the work we did in natural products/metabolomics comes from neurobiologists, virologists or biochemists. They do not have NMRs in their labs or would even know how to run/select/interpret the experiments.

...1) the 'quantity' of the samples for single crystal X-ray diffraction are far-far less than those for NMR. Especially for heavy nucleus determinations. For small molecule analysis it is need ony one crystal with size 50-200 microns. Such 'amount' dissolved and analysed via standard NMR techniques does not give any 1H-NMR signal (personally tested on 400 MHz instrument). Really has very valuable studies of the 3D structures of biological macromolecules, achieved by NMR, but....again the method is not 'absolute', and cannot provide information for the 'absolute position' of the atoms in the structure, which is exactly the capacity of single crystal X-ray diffraction (small or protein).

....2) for SAR analysis, metabolomics, neuroscience, medicinal chemistry and related the MS is exactly among the most powerful methods as well, allowing direct in vitro and in vivo assay and imaging (MALDI method), including on tissues and whole organs. In addition, the concentration limits of detection (LODs) of analytes is up to fmol-attomol ranges achieved by MS methods. Such LODs are generally out of the capacity of NMR. Because of NMR is scarce utilized for quantitative determination and it is very difficult to find in the literature a meaningfull quantitative anaysis with chemometrics, which to illustrate the real boundaries of its applicability for quantitation of small amounts of analytes in complex biological and/or environmental matrixes (tissues for example). Furthermore the sample pretreatment steps in NMR are more complicated, time and labour costing ones than those utilized in the MS bioassay.

I fully support crystal X-ray diffraction studies but limiting factor is single pure crystal :-)

There are pros and cons of NMR spectroscopy and X-ray techniques.

Considering the complex matrix of natural product samples, this fact alone is one hindrance of achieving excellent purity of natural product samples that would suffice for the isolate/chemical entity to become crystalline in form. Apart from that, there are many isolated pure compounds from natural sources that are always amorphous in form, no matter what sample processing/purification is done. With amorphous isolates, there are no crystals, so there is no possibility for X-ray determinations. It would have been ideal if all chemical entities would exist as crystals for that matter that so we can always resort to X-ray techniques and take advantage of the sensitivity of the method. But clearly this ideal setting is not always true, so alternative methods for structure elucidation are needed to complement X-ray crystallography techniques.

On one hand, NMR techniques undeniably will always have a stronghold in the field of Natural Products, Organic, Medicinal Chemistry and Biochemistry , historically and for practical reasons. The rich and growing database of NMR data serves well to clearly identify and dereplicate known compounds, ranging from small molecules to complex structures like proteins. Furthermore, the advent of multi-dimensional NMR analysis paved the way for comprehensive and thorough analysis of novel molecular structures, small or big. In proteomics, some proteins can be crystallized and some others cannot be crystallized. In the least there are proteins that are not possible for crystallization within the limits of a research method. In this case, multi-dimensional NMR elucidation is employed for those proteins that cannot be prepared as crystals. And also significantly, for reaction intermediates and specific chemical entities that can only exist in solution, NMR is the only comprehensible method to use for their structural characterization. These scenarios are but a few of the instances where we realize the power and importance of NMR as an indispensable tool for structure elucidation. Not to mention the fact that structure elucidation is possible even for mixtures. Notably for mixtures consisting of combination of isomers or very closely related molecules (structures differing only slightly in selected moieties) , NMR proves really indispensable. For Natural Products Chemistry in particular, especially those concerning isolation studies of plant metabolites, isolates are usually less than pure. More often than not, isolates consist of a mixture of isomers and/or very closely related molecules which can be very difficult to resolve. For experienced NMR spectroscopists however, they may be able to fully elucidate the structures of the individual molecules in the mixture of compounds despite the overlap of individual NMR spectra. By peak intensity ratio analysis of the NMR signals, it is also possible for them to estimate the proportion by abundance of the individual compounds in the mixture. The analytic power of NMR comes with a cost, however, and that is more quantity of sample is required to obtain a good NMR readout when compared to X-ray determinations.

In essence, both NMR and X-ray techniques are useful in the process of structure elucidation. Each has its own practicability and specific applicability in a case to case basis. But of course just as in in any scientific endeavor, it would be better if both X-ray and NMR measurements can be done on a sample so that both sets of experimental data can counter check each other, as this is the usual practice in Organometallics. Let us not forget also that the more evidence that can be produced to justify the structure of a molecule, the better it is for the elucidation study. Hence it is worth mentioning that, whenever possible, backup experimental determinations like MS, IR, Raman, UV and CD analysis as well as physicochemical determinations (e.g.: melting point and boiling point measurements) should be done to further explain the structure of a compound. A holistic presentation of various experimental data to justify a molecular structure would make an excellent material for a research paper.

My version of 'Murphy's Law' for structure elucidation is that, if it is difficult to interpret the NMR spectra of a molecule because it is impure or it is isundergoing conformational averaging at an intermediate rate, it probably won't crystallize. Higher field NMR spectrometers can actually be a disadvantage, particularly for 13C spectra, in the latter situation. Because of the larger chemical shift differences (in frequencies), 13C peaks can actually become so broad that they can be lost in the noise. However, high field is almost always better for 1H NMR. As far as sample requirements are concerned, it is certainly possible with a He-cooled probe or a capillary probe to carry out 2D NMR for structure elucidation with less than 1 mg of sample. Even if crystallography can be carried out with even smaller amounts, this is basically irrelevant for most natural product research because it is next to impossible to get the work published in leading journals if one can't isolate sufficient sample to carry out at least some bioassay tests.

To come back to the original question which was about couplings at different fields. The coupling constants are of course field independent. The chemical shift (in Hz) is not. This can change the appearance of the spectra. If you suspect long range coupling I would run a simple TOCSY with a mixing time >60ms. This will tell you what is coupled. and what not.

As an aside, just because something appears as a singlet it does not mean that there is no coupling. Consider adenosine, the both H2 and H8 protons appear as a singlet…. but they are in fact coupled (ca 0.3-0.5Hz). A long range Tocsy can show that. In this case we are talking about a 7/8 bond coupling …. which is rather remarkable., and you can even see this in DNA duplexes… once you know how to look for it.

See also Jin Zhang et al. JACS 2009, 131, 5380.

I appreciate that valuable piece of information Dr. Germann.

I also have accessed the article from the ACS journal archives.

I fully agree with the answer given by Markus. May I add a comment? With your high field NMR spectrometer, not only you gain a huge gap in sensitivity, due to the increase in difference of populations between the alpha (parallel) and bêta (anti-parallel) spin states (this difference builds up the signal/noise ratio), but also, in addition to COSY-LR (TOCSY, HOHAHA = Homonuclear Hartmann-Hahn Spectroscopy, ...) as Markus told you, it allows you to record 2D HETEROnuclear NMR spectra : the HSQC = 1J-1H-13C correlation, from which you get all the protonated carbon spin systems and the HMBC = 2,3,4J-1H-13C correlation, allowing you to link all those spin systems and thus, to construct the only possible structure! Since these spectra are recorded in the "reverse" mode, that is to say, you record proton spectra (at the sensitivity of the proton, ±5700 higher than the 13C spectra!, and at the huge resolution of the proton), which are modulated by the frequency of the correlated carbon atoms in the virtual dimension, you are able much more easily to figure out the framework of most of the soluble metabolites, and to deduce the right structure, which then you compare with the already published data, if it is known. Sometimes, these spectra push you to assign a correct structure to a misinterpreted old one.

If the TOCSY (with a quite long mixing time as suggested by Markus is giving you a deep insight into the proton spin systems, you have then the opportunity to transpose these results into an heteronuclear 2D NMR spectrum (HSQC-HOHAHA!).

It is just a matter of knowledge in setting up the conditions for each experiment, but as they do not last a long time (a few minutes, for each of them) you should try as many conditions as you can, to discover the truth.

Good luck

Joseph

While the 1H-detected heteronuclear correlation sequences (e.g., HSQC and HMBC) are more sensitive than their 13C-detected counterparts (by about a factor of 16 on average), in cases of spectral crowding, there are resolution advantages to using the older sequences (HETCOR and COLOC or FLOCK). One can improve the resolution along the detected axis, f2, by adding additional points at little extra cost in time, but doubling the number of f1 increment spectra double the time. Thus f2 data point resolution is better long f2 than f1. since the 13C spectral width in frequency is typically ca. 5 times that of the 1H spectral width, one can get better resolution by having the wider 13C window as the acquisition axis.

I do agree, all these high frequency NMRS are very good and give huge information about an unknown compound. But, sometimes too much information also make things complicated!!! These secondary couplings/long range coupling at high resolution make spectra complicated.

Dear Zakariya,

The higher field increases the signal separation and the sensitivity.

I send you a picture of spectra recorded on different field attached to illustrate it.

See the answer of Marcus W. Germann!

Best regards,

Sándor

The coupling constant, chemical shift constant, and shielding constant would not be dependent on the magnetic field strength, but the chemical shifts would be, so as the C value and the ratio intensities.