Nuclear Magnetic Resonance - Science method

Hi William C Hutton ,

Thank you for your answer. Unfortunately, due to circumstance I cannot do any more experiments now (otherwise I was thinking of using D2O already).

What I have observed is that I have got splitting of some of the acidic sites into a 2:1 doublet or an asymmetric but very well-resolved triplet.

I have re-admitted air into the sample rotor step-by-step and measured a few times. The (sharpening and) splitting has come progressively with more exposure to air and has passed through a max. splitting. The magnitude of splitting is 60 to 70 Hz. We're talking about isolated OH groups (no or little H bonding involved), I've very sharp lines for solid-state NMR.

For instance, an initially 2.73 ppm resonances gradually splits into 2.85, 2.72 and 2.60 ppm. There is slight shift of position.

The basic OH group at very high-field does not undergo splitting, supporting the assumption of proton exchange at the acidic OH sites.

But I cannot understand why such a splitting occurs if it is simply the protons being exchange between H2O in the immediate surrounding and the OH at the surface.

The sample is gamma-alumina. My first theory was actually something completely different: namely, the asymmetric triplet is caused by a residual dipole-dipole interaction between 1H and 27Al (1/2 vs 1/2, 1/2 vs 3/2, 1/2 vs 5/2). But the way the splitting occurs (i.e. increases by increasing humidity exposure) makes me a bit skeptical about this theory.

So based on the above details, what would you assume?

Forgot to attach the code....

Both VT-HT and VT-LT are available at SAIF, IIT-M for external users

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Luc is perfectly right. I REALLY depends very much on what you want to do. To add two which is widely used for quantitative NMR based (plant) metabonimcs: https://www.chenomx.com/ or https://spin.ccic.osu.edu/index.php/colmar

... but is definitely is NOT the one if you are after de-novo structure determination of yet unknown natural products (here ACD with all its packages may help, as Luc already pointed out).

Dear Lorenzo,

I am VERY much wondering why HSQC is a problem.

Doing so you can estimate how many more scans it will take to have a signal substantially higher than noise.

All this is of course ONLY true for small molecules where relaxation can be neglected.

In our experience matching and tuning is NOT highly affected on 13C by high salt....

Goog luck

Alfred

I can't help you with your purification or reference spectra, but we do occasionally work with comparable lipids and they tend to dissolve relatively well in methanol/chloroform mixtures.

I don't think your question is specific enough. Where is this extra peak located for instance. What is your NMR solvent? And if you are doing a basic ester hydrolysis, what do you mean with "hydrolysis in acid NMR"?

you should convert the alcoholic endgroups into esters by reaction with (non proton bearing) derivatisation reagents like trichloroacetic anhydrid or trichloroacetyl isocyanate. See: Polymer Bulletin 1991 , 27, 201-204

Best regards and good luck

Gerhard

You should only be getting one peak. What is the ratio of the peaks?

Could the other peak be residual glycolide?

Does the CHCl3 peak look normal, or could the two peaks be caused by poor shimming?

Can you run a 13C spectrum to verify that you actually have two peaks?

Thank you Gregory J Rees!

Dear Paul,

William gave an excellent explanation...nonetheless three questions:

* Are ALL peaks in 2D (presumably 1H-15N HSQC?) affected or only a subset (in line with Williams explanation).

* How do 1D 1H (e.g. watergate) with long interscan delays (>10s) of the two samples compare?

* Have you deterined the 90° pulses for both samples? Absence or presence of salt (coming or not comping with the protein constructs) may be another explanation.

Alfred

it depends on the MW. If you can see the resonances of terminal units ( usually in polymers they have a slightly different Chemical shift) you can try to make the calculation of MW from the ratio of their integral with respect to that of the CH2 units of the polymer , Otherwise a DOSY 2D experiment may give an estimate of the molecular Diffusion from which you can derive the MW by the Stokers Einstein equation.

Hi Mathilde. This is perfectly normal. The molecular weight obtained by SEC is not actually molecular weight. The SEC instrument 'sees' the hydrodynamic volume of your polymer and reports the molecular weight relative to the hydrodynamic volume of standards that were used to calibrate the instrument. So you will always get different results when you compare molecular weights obtained from NMR and SEC. Hope this helps.

For a 1H-31P bond there will be a J-coupling, however as others have noted it will typically not be irectly observable due to the more dominant dipolar coupling (through-space).

It might be possible to measure the coupling using spin-echo based methods as shown in https://doi.org/10.1016/j.crci.2009.05.001

It is helpful to execute this command while another data set is open. Otherwise there are problems with stoing the converted data set. if the jdf file is digitally filtered data use PHC1 of ~ 6900

You can use POKY for that (https://poky.clas.ucdenver.edu).

Poky menu -> Universal NMR Formats -> NEF chemical shifts to Sparky resonance list

Once a resonance list file is generated, it can be loaded in the project from the resonances list tab or from the resonance window (rl).

Then, you can repopulate assigned peaks using Transfer-and-Simulate tool (ta).

You can address the Effect of Solid Lipid Nanoparticle Incorporation on the Lipid Digestion Kinetics and Bioaccessibility of Encapsulated Bioactive Compounds in Food Grade Nanostructured Lipid Carriers: A NMR, XRD, and DSC Study.

Dear Nuruzzaman,

If the Educt has also a 19F you do not need a reference, because the ratio of the 19F signals of educt and product is the yield. NMR signals directly represent molar ratios. For 19F you may have to take care for the excitation profile of the pulse you are using. I normally place the carrier in the middle between the educt and product signal (doing so, both have the same offset effect)...

I hope this is of help!

Alfred

I suspect this issue is related to the gradients. I would decrease the gradient strength and, or the delay after the gradient(s) to see if the resonance shape near the baseline becomes more symmetrical.

Residual vibrations caused by eddy currents contain anharmonic vibrations. The discrete FT requires the decay of all time-domain signals to be pure exponentials. But residual eddy current vibrations contain components that decay with with nonharmonic, nonperiodic modulation. The DFT can not accurately model these anharmonic signals. The problem isn’t the eddy currents, it’s that the DFT mathematically processes the anharmonic components as if they were harmonics.

The result is often an artifact in the wings of the frequency domain water-signal baseline similar to the one in your figure. Details about the impact of nonharmonic, nonperiodic water signal components can be found here: “High dynamic-range magnetic resonance spectroscopy (MRS) time-domain signal analysis.” Hutton, W. C.; Bretthorst, G. L.; Garbow, J. R.; Ackerman, J. J., Magn Reson Med, (2009) 62, pp 1026-35.

Jani Rahkila one of them is 975 g/mol and the other one is 328 g/mol.

No, I don't have access to the NMR machine at the moment.

Dear Paul,

essentially all explained by the McConnel equation https://groups.chem.ubc.ca/straus/Nlecture3.pdf

Alfred

I'm not sure if this answer applies to ¹⁵N HMQC experiments, but in the past when we did 2-D NMR experiments to measure ligand binding to a protein by measuring chemical shift changes of peaks, a loss of multiple peak intensities was taken to mean that the ligand was binding in a non-specific manner, such as an aggregator or denaturant. Such compounds were discarded.

The magnet itself does not produce a significant amount of heat, but the console cabinet with all the electronics does.

Your local Bruker office (https://www.bruker.com/en/services/support/office.india-middle-east-africa.egypt-gizeh.html?technology=magnetic-resonance) can provide you a site-planning guide (or you can find one via google) with all the information you need.

Robin is correct. The Schiff base is always an equilibrium and must be reduced (e.g., cyanoborohydrin) to create a permanent imine. You have to use an aprotic solvent (e.g., acetonitrile) to trap the Schiff base. There is also a byproduct urea that forms that is 1 Da off in mass.

Without knowing the specific structures of the two chemicals in question, it is impossible to predict the exact positions of the 13C NMR peaks. The position of 13C NMR peaks depends on the unique chemical environment surrounding each carbon atom in the molecule, which is determined by the arrangement of other atoms, the type of functional groups present, and the hybridization of the carbon atom.

However, as a general guide, here are some typical 13C NMR chemical shift ranges for various types of carbon atoms in organic compounds:

It should be noted that these are only general trends and chemical shifts may vary depending on the specific compound and the context of the carbon atom in question. A detailed interpretation of 13C NMR spectra requires careful analysis of the entire spectrum, as well as knowledge of the specific chemical structures being analyzed.

1)answer 1: first time, please obtained FT-IR of the monomer (which monomer of your obtained polymer), and then In FT-IR, the presence of a peak around 1700-1750 cm-1 can indicate the formation of an ester bond. This peak corresponds to the carbonyl stretching vibration of the C=O bond in the ester group. Additionally, you can also look for a peak around 1100-1050-1100-1300 cm-1, which corresponds to the C-O-C stretching vibration in the ester group.

2)n FT-IR, the presence of a peak around 1600-1700 cm-1 can indicate the formation of a C=N bond, carbonyl bond. This peak corresponds to the stretching vibration of the C=N bond in the imine group. Additionally, you can also look for a peak around 3300-3500 cm-1 screeching and Overton of this, 1400- 1500- 1450 cm-1, which corresponds to the N-H stretching vibration in the imine group.

Verkade base is one of the strongest base; after protonation, it forms a conjugate acid with pKa value of ~33. That means it can deprotonate even a weakest acid and hence the signal disappears. This includes for your both the questions 1 and 2. Conjugate acid is = [HP......]⁺ [pKa = 33]

Best

Balakrishna

UCSF is the spectrum format in frequency domain. You can process with TopSpin to generate a 2rr/3rrr file, and you just convert using POKY.

Watch: https://youtu.be/ePxKeKaIzpE

Hi, a crude measure is to use -ignh during pdb2gmx, it will rebuild all the H-atoms based on the force field you are using. Most of the time, it is a reasonable choice (though not always), as the H-atoms are mostly absent in crystallographic structure (as it is difficult to resolve h-atom positions).

Histidine is unique in the sense that its side chain offers multiple h-bonds at physiological pH. The better procedure is to check which heavy atom of His side chain is forming H-bond in the protein (either it is delta or epsilon), and rename your His residues accordingly (HIS, HIE, HID, please check your FF how these residues are named there).

"2. I do not know if I should add the atom HD1 in the HSE entry in the rtp file (I tried this and got several warnings)." > Try not to mess with rtp entry at this stage (as you are very new), and if you like to play around, just make a backup of FF directory and do as you like.

Hello Saya Bird,

the purification depends on the polymer and the side products/impurities, which you want to remove.

Additional purification methods might be following:

Dialysis; I used this to remove some salts, which could leave the polymer solution through the membrane of the dialysis tube, whereas the polymer remained in the tube.

Fractionated precipitation: I used this to separate blockcopolymers from homopolymers or different moecular weight fractions of the same polymer from each other. You add a non solvent to a polymer solution and the less soluble polymer (depending on their chemistry in the case of block copolymer) or the polymer with the higher molecular weight (in the case of different molecular weights) precipitates first as a polymer gel.

There are also methods, that require more elaborate equipment, like preparative GPC or separation by LCCC.

If your separation does not remove the impurities, maybe add the solvent slower (drop by drop, as you mentioned) and use another solvent (or non-solvent). Maybe you polymer precipitates so easily, that the impurities are trapped in your solid polymer. If the polymer precipitates slower the impurities may stay dissolved (similar to the case of fractionated precipitation).

The confirmation of your product also depends on the polymer. Maybe some groups on your monomer were changed by the polymerization, which could be detected by IR-spectroscopy.

For the chracterization of polyurethanes (as an example), remaining isocyanate groups can be titrated.

If you use a new method to obtain a known polymer, you can also compare it's properties with literature values (i.e. the glass transition temperature via DSC) to get a hint if the reaction might have worked.

There are plenty of complex and simple methods to characterize a polymer, but it depends on your polymer if you can use them.

- ERETIC2 and QUANTAS are both software approaches based on the PULCON method of Wider et al. (J Am Chem Soc. 2006 Mar 1;128(8):2571-6. doi: 10.1021/ja055336t). Note that the original ERETIC (Akoka et al.) is a hardware-based approach and may not be suitable. QUANTAS reference: Magn Reson Chem. 2010 Oct;48(10):753-62. doi: 10.1002/mrc.2647. PMID: 20803488.

There is no reason why these approaches can't be used in a benchtop NMR.

tes

See this paper

Dear William,

Here is a good website explaining the T1 dependence with magnetic field and including references.

It focuses on 1H, but gives a good general idea.

Best,

Maxime

Solution = iNMR

Since the molecular weights of the components of your mixture are significantly different, you may try 19F DOSY to distinguish the 19F signals of CF3 groups. See, for example, https://pubs.rsc.org/en/content/articlelanding/2016/cc/c6cc02917e

To complement Clemens answer, I would like to mention backward linear prediction, which is a standard procedure in almost all NMR processing software packages. It helped me a lot for measuring 11B NMR spectra using standard (non-quartz) NMR tubes.

Good luck,

Vladimir

In my suggestion the title may be " Characterization of Food materials for their quality"

Dear Alisha,

to answer the question I (we) need to know the reaction-scheme.

Was it a trans-esterification of glycerol-esters? If this is the case I'd guess that the signal at 3.6 is the O-CH3 of the product (which you have divide by 3, as described above) and the signal at 5.2 might be one hydrogen of the glycerol in the educt. And I(O-CH3)/3/I(educt) would be the molar ratio of product/educt (which is not the necessarily the yield!). But this might be wrong if my "hand-wavy" assignment is wrong.

Alfred

Do you have a Scifinder or reaxys access? They both have very good structure and substructure search masks, so if there is a mass spectrum in a good journal, they should hopefully find it.

Dear Prasad,

Cu2+ usually increase T1 relaxation but not really T2 relaxation (which would be bad). This means you can reduce the D1 delay to reduce measurementtime. Otherwise it should be fine to record 1H experiments, but no garantee. to determine the T1 relaxation and teherefore the d1 delay time you can use the parameterset 1HsatrecT1. after recording you can click on "Applications->Dynamics->Prepare for Dynamics Center" and than again at the same button Saying "Dynamics Center" and the result should pop up. If you have installed the free of charge Bruker Dynamics Center on yur computer.

Many Greetings,

Kristof

Dear Ahmad,

Thank you for your detailed answer, I eventually managed to solve the problem with the aid of Bruker technician. The problem was due to an insufficient cooling because of a bad vaccum in the liquid nitrogen transfer line, I just had to redo it.

Many thanks.

Salem

It depends what level of accuracy you require. In simple terms, identify a signal from your product and one from your reactant, integrate and normalise these for the number of protons that they represent and these numbers will give you the molar ratios of the product and reactant. There is no need for an internal standard in this case.

There are caveats. This method assumes that your reactant only produces product and that there are no by-products. If there are by products then you could also integrate signals from these. Alternatively, use a reference signal such as ERETIC2 or QUANTAS and this will allow you to quantify independently.

If you require accurate results, then you need to follow William C Hutton's comments about signal-to-noise, etc. - particularly relaxation delays!

The time domain quantification of metabolite signals was conducted using AMARES algorithm with custom prior knowledge.Metabolite concentrations were reported for tCr (creatine plus phosphocreatine), NAA (N-acetyl-aspartate), tCho (phosphocholine and glycerophosphocholine), Ins (myoinositol) and Glx (glutamate and glutamine). The AMARES prior knowledge model consisted of peaks for NAA, choline (Cho), creatine (Cr), glutamate + glutamine (Glx) and myoinositol (Ins). The amplitudes of NAA, Cho, Cr, Glx, and Ins peak were estimated by the algorithm. The relative phases of NAA, Cho, Cr, Glx, and Ins peak were fixed at 0. The linewidth of NAA was estimated by the algorithm, and the linewidths of the remaining peaks were set to be equal to that of NAA. The frequencies of NAA, Cho, Cr, Glx, and Ins peak were estimated by AMARES. All peak shapes were fixed at Lorentzian.

you can use CF3COOD/CDCl3 (2/1 v/v)

Dear David Stephenson, Thank you for the tips. They worked.

The problem has been solved. Thank you for your professional reply

Abel J S C Vieira Henri Strub Alfred Ross Tabrez Shaikh

Thanks a lot for your precious suggestions.

Nitika Grover hope this helps.

US5116997A - Purification of indigo - Google Patents

Dear Omar,

Interesting question....I never thought about it. If once the cool-down is done (only done once while installation of the machine) the calculation for "regular operation" can probably done like this:

Latent Heat of Evaporation of N2 is 200 kJ/kg. If we assume a loss of 100 l per week (actually it is lower for a 300 MHz machine) we end up if I calculated correctly about "30 W" of "cooling power" 200.000[kJ/kg]*0.8[kg/l]*100[l/week]/(24[h]*7[days]*3600[s])..I'll guess the He contribution can be neglected, but the electronics of the console adds a few hundred watts.

So at the end for a decent air-conditioning no problem!

I hope I calculated half-way correctly

Alfred

Dear Lubna Afroz a conclusive analysis and answer to your question can only be given if the splitting pattern of your signals is clearly perceptible. This is not the case with your illustration. Swapnamoy Ganguly , I do not agree with your analysis for two reasons: (1) you cannot be sure that the signals are "triplets" or "quartets" (I doubt about that) and hence the calculation is questionable; (2) coupling constants larger than 20 Hz are very rare, not to say unlikely for organic compounds.

Moreover, applying a line broadenig factor of 1 Hz is not really helpful for most proton spectra, and the integral labels look very strange to me (as there are four regions of relative intensity 2 but only three peaks groups of appropriate size). Please check everything.

As John mentioned, it depends. Mostly because molar concentrations of the active nuclei matter in spectroscopy, not the w/w or w/v. So, 0.1 mg/ml may be pretty ok for 1H NMR of a small molecule, but not the large one and not (natural abundance) 13C NMR.

A stable derivative of the compound is made using a chemical like TMS or equivalent which then will not undergo further decomposition during the chromatography step (eg. ). Alternatively, you could quantify the increasing degradation products and infer the remaining amount of parent compound.

Dear Fatima,

without a clear terminal (or close-to-terminal as Clemens mentioned) signal with a defined number of atoms per polymer contributing to the NMR signal it is not possible!

The integral of a NMR signal is given by

(molar concentration of a polymer)*(number of atoms in this polymer)

Thus half of the molar concentration of the polymer with twice the number of monomers unit (double MW) will lead to the same integral.

An estimate of MW may be obtained by measuring diffusion (DOSY technique) coefficient D. But you will need (several) reference polymers of same type/chemistry and known MWs for calibration

...or you find something useful in literature.

A rough estimate of the MW may be "guessed" if you only have one MW reference by a polymer-scaling law - to my remembering D(MW)~MW^(3/5) for unstructured polymers. But please check this!

If you have MW references there are more simple (SEC, gel-filtration) chromatography methods.

Good luck

Alfred

Wolfgang's answer is absolutely correct in every respect. That said, I still struggle with alternatives to upfield and downfield. Try putting the alternatives into a descriptive sentence. You can't very well say that a signal is "higher" than another signal as this could refer to its intensity. You could say that it is "higher frequency" than another signal but the chemical shift scale is dimensionless (it is normally described in "ppm") so this perhaps isn't truly valid either unless your scale is in Hz. I've been told that "to higher ppm" or "to lower ppm" is valid but it is ungainly and counter-intuitive as people tend to thing of higher being to the right and lower to the left (which is the opposite of the delta ppm scale). Perhaps we should go back to tau where TMS comes at 10ppm... (only joking).

Der Mashid,

not any COF is soluble. Solid-state NMR might be an option. For soluble ones also mixtures of ACN with water or acidified water was reported...

Yes there are, if you are lucky then may be you will find exactly the answer on the possible reactions you are dealing with. Please have a look at the following links. My Regards

generally if it happens in DMSO-d6, one can heat the sample with heating gun or else after adding D2O some compounds will get dissolved.

Hallo,

It's not a big difference if the polymers are different or the concentration is different. What is the standard deviation of these coefficients you mention? The polymer signals may have a good decay and with enough increments for the fitting, this may not be the case for CDCl3. Do you have a good decay for the CDCl3 signal?

You could repeat the experiments with the shortest possible d20, the diffusion coefficient of the components in the sample should be "equal" to your first measurement.

If you are using an edited pulse sequence, mestrenova will not be able to calculate the diffusion time (big delta) properly and this results in an error.

Any difficulty look for our group in Regensburg, we can help.

Apparently 2,4-di-tert-butylphenol is produced by dozens of different organisms. I am still in two minds as to whether this is true, or the result of either environmental contamination or bad lab technique. The more I read on this topic the more it seems it is indeed a natural product. It could be easily resolved by conducting an experiment where 13C-glucose is used as the sole carbon source...

I greatly appreciate all the suggestions. Thank you.

In this paper Authors didn't provide NMR data. Many publications sited this publication about this Emblicanin A and B content but none of them provide NMR data. Thank you.

It is not clear from the question what the reviewer is demanding. What kind of calibration? Probably calibration of the chemical shift. This is a very tricky thing and a lot of discussion has been on this problem. An external standard for this purpose always performs inferior compared to an internal one and external referencing is not recommended any longer, therefore. See, for example, Rosenau et al. Angewandte Chemie 2018 (doi: 10.1002/anie.201802620). My personal recommendation is to make a clear and unambiguous statement on the referencing procedure. It is not sufficient to state "the solvent/water signal was used as reference", it is better to write "for chemical shift calibration, the water signal was set to 4.71 ppm" or something like that. This is clear, understandable and unambiguous and will assure for 100% reproducibility.

Dear Wu Chuan,

the chemical shift of the residual D2O (actually HDO) does depend on the pH of the sample. You may guess that this pH does not vary if poly-saccharides are dissolved (which will likely be true, if the molecules so not have chemical modifications)....

I hope this helps

Kind regards

Alfred

Dear Meltem,

I guess that you already know this literature:

Good luck

Alfred

Thank you for your answers.

Haitham Saad Al-mashriqi

Afroza Begum

Jun Uzawa

For interpretation purposes there is no difference. The difference is in the acquisition process. Depending on the device and software used in spectra processing(MestreNova) , in HSQC it is possible to distinguish the CH2 carbons. Important to highlight that both H-C cosy or HSQC, the observed correlation is C-H.

Ganguly

1.4 Tesla is the maximum field strength achievable in a conventional configuration (natural magnet-at the time) which was known in the early stage of NMR development (before the manufacture of superconducting magnets).

Good luck

For a tightly binding small molecule you could diffusion filter the spectrum. Bound drug should have a significantly different diffusion constant compared to free.

James E Hanson Thank you very much for your valuable help

The following link is also very useful: https://www.googleadservices.com/pagead/aclk?sa=L&ai=DChcSEwj3zfWbsen3AhUYp3cKHRSqDcIYABAAGgJlZg&ae=2&ohost=www.google.com&cid=CAESbOD2KZ40kaCxmGfg3ASL3Jzer14yFfMtRQpRN7fxEU9NOMowXRZT-1uO8b3T1HHdtrtl8rw9Afav-wg959rbQPDwAksU-qmO-K5w97g0ejz124AzSUR7ji_XNGiOHYsai6JRszlD0zYt_Q-GFw&sig=AOD64_0Ss0AIrwAyKttE-v5aARo_n6GVaw&q&adurl&ved=2ahUKEwiTpu2bsen3AhV3iv0HHZ5wALI4ChDRDHoECAAQAQ

Dear Sir,

If your compound has low molecular weight and has volatile nature, you can detect the compounds by simple GC-MS or headspace GC-MS.

When re-reading your question I see the phrase "....does solvent (DMF. EtOAc and DMSO) which is used for reaction and workup affects percentage yield...".

Based on this I conclude that your question actually is NOT a NMR question, but a synthesis question. With my very limited synthesis understanding it is clear that different solvents will induce different reaction kinetics related to differences in yield....

I hope this helps

Alfred

P.S. Thanks John for pointing out the 13C satellite problem! This sometimes is important to remember!

I see so many of these questions! Is there software that will solve the answer to complex NMR data? The answer is "no". There is software that will process NMR data, software that will attempt(!) to confirm a simple chemical structure, and software that will aid assignment.

Fundamentally, especially for something as complex as polysaccharides, there is no alternative to an expert.