Here is an article dealing with this subject:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993187/

Hi Casas,

You can use H3PO4.

regards,

Uday.

Thank you Adam and Udaya. Probably my best option is to use an internal standard dissolved in D2O (for locking purposes) within a sealed capillary tube within my experimental samples. The internal standard can't be orthophosphate (as Udaya suggested) as my experimental substrates and products can easily generate it by hydrolisis, and thus this would interfere with the quantification purpose.

I guess I need to find another phosphorus-containing molecule that has 31P-NMR peaks not interfereing with those of AMP, ADP, ATP and acetyl phosphate (my reagents/products). And also, this IS should be rather stable and not degrade quickly in solution with water. Any molecule suggestions?

How about trimethylphosphate? or may be triphenylphosphine is a good option.

Thanks Udaya. NMR shift calculator predicts the peaks won't overlap for any of those, but I guess I'll have to buy them and see their real spectra.

Trimethylphosphate is soluble in water (or D2O) so might be the best option; I hope its real peak doesn't overlap with the orthophosphate peak which will undoubltedly appear in my experiments.

Triphenylphosphine is soluble in organic solvents only: would there be quantification problem if my analytes I are dissolved in water and the IS (triphenylphosphine) whithin the sealed capillary tube is not? I guess I could still add 10% D2O to the sample for locking purposes anyway.

Are both of these molecules rather stable? I guess I could prepare a fresh IS solution once a week and keep it at 4C, probably that would suffice.

 Actually I also have some methylphosphonic acid which should have a very strong shift (around 29 ppm) so I think it's worth giving it a shot too.

How about TCEP (tris(2-carboxyethyl)phosphine)?

Hi Eloi...methylphosphonic acid should serve the purpose.

If you consider PPh3, you can simply use a capillary of organic solvent. PPh3 should be available (I would say in 100s of grams) in any organic chemistry lab.

I concur with UDaya. Seal a capillary tube with an internal standard, center the capillary tube inside the NMR tube as your internal standard.  One would need to do three NMR runs for calibration.  1) surround the capillary tube with a blank solvent,  2) replace blank with a sample of known concentration of P compound, and 3) replace standard with unknown P compound.  Need to validate the precision of the procedure. The field of view of the compound in the capillary may not be [precisely] identical to the field of view of the sample [one could be seeing a higher or lower mole fraction of the IS than of the sample of interest].

 Thus the need to calibrate the concentration inside the capillary versus a known compound.

P compounds that have sharp singlet NMR peaks could also be chemically reactive, when most often, one wants an internal standard that is chemically inert to one's compound of interest.

Thanks Walter. I will most probably use methylphosphonic acid disolved in D2O inside a sealed capillary tube as internal standard. The P molecules I want to quantify are, for example, ATP. Following your advice, would it be: 1) lets say make a dilution of 10 mM methylphosphonic acid in D2O and seal it inside capillary tubes (I hope it's fairly stable and I can use one tube for a few weeks). Then just add normal water as a blank solvent, and measure the peak intensity. 2) Then replace back with a sample of known concentration (for example 1 mM ATP) and measure it. 3) Do the normal experiments with unknown ammounts of ATP.

Is this what you meant?

Would I need to do this everytime I submit samples or just once at the beggining of the project?

Thanks!

Yes, you are correct.  Also are correct about the molarity of the IS and of the ATP best are similar.   Assume both the IS and the primary standards of known conc ATP are equal.   One can thinks one measuring concentration in the NMR tube, except that is not true.  One could place the sample in a sphere and fine tune the z direction to exactly the sweet spot of the NMR and the signal would be identical with that of a cylinder of the same diameter containing the same concentration. 

Said another way, if one uses a wider bore capillary, the relative concentration if the IS will be higher and the relative volume of the primary standard will be lower.

The blank will let you know the NMR scale required for measuring the relative IS concentration.

Again, if inside and outside the capillary have equal concentrations, the only variable [reason the signal would not be equal] is the relative volume of each detected by the NMR.  Only if the volume in the capillary cylinder versus the volume of the sphere not in the cylinder were equal would there be no volume ratio correction factor.

As long as the volume fraction in the cylinder is a constant [and the NMR and NMR shimming are essentially constant], the IS contribution to the signal determined by the NMR will be a constant.   You can prove this to yourself just by rerunning the 1mM ATP later and seeing how close to the last time the signal relative to IS was.to the first time.

Organophosphates I think are quite stable compounds.  If there were any IS chemical breakdown / rearrangement, almost always that would show up in the NMR itself, especially in the blank run: would no longer be a singlet at a single frequency.  A 1% degradation would result in a 1% peak at another frequency, probably also a singlet.

Thanks Walter. If we were to spike our samples with a known concentration of internal standard (not inside a sealed insert), then I guess that the areas of analyte and IS would be directly comparable, right? The only possible problem would be that the IS and the analyte/reaction substrates react.

Also, another way of quantifying ATP could be to just use a normal calibration curve generated with different known ammounts of analyte, right?

Exactly.    Just like one mole  of  protons on a CH3 site  integrate for three proton whereas a -CH2-O  would integrate for two, it make zero difference whether the protons are on the same or a different molecular species.  Of course you need to know the number of moles of protons are on each peak of interest.  Otherwise the math can be correct and the answer wrong because one got the chemistry wrong.

The best internal standard is in region where there are few or even no other NMR peaks in your sample matrix.

Yes, can also do a standard curve.  With high field NMR,  an IS in practice does not improve accuracy much. Did quantitative NMR on even a 60MHz NMR, and the major reason for an IS was that integrations were made six times and averaged.  The magnet was not stable or uniform enough otherwise do get a good enough result.

Now virtually no one does an peak integration more than once.   One can test whether the integration is accurate enough by running ethyl acetate. Unless the peak ratios are  within  2-5% of the proper ratio,  cannot expect an assay in a mixture to better than 2-5% of the right answer. .    .