Thanks everyone for your help in finding a 31P reference compound. In my
original query, I was looking for a reference compound for our 31P studies
of bacteria which would not interfere with the compounds of interest.
Below is the summary of the responses:
When I analyze phospholipids mixture by 31P, I use triphenylphosphine. Using a
solvent ratio 1:3 of Methanol/CDCl3, relative to H3PO4, shows its signal at a
chemical shfit of -5.29 ppm.
The standard for 31P NMR is external 85% H3PO4 in H2O. Beware of bulk
magnetization effects. Concentration affects the chemical shift so if you
want accuracies better than +/- 0.05 ppm, check the concetration by titration.
Two secondary references were suggested: eternal hexamethylphospharimid
in H2O (30.73 ppm) and internal dilute trimethyl phosphate (3.09 ppm CDCl3,
3.78 ppm D2O, 3.49 ppm CD3OD, 3.95 ppm acetone-d6).
We have used MDPA for years in our perfusion studies with the eye. It
was sealed in a capillary and put directly in the perfusion cell. In
our calibration studies with phosphoric acid set to 0.0ppm, MDPA was
referenced to 18.0ppm, out of the way of anything of interest.
I think that if you want some standards try these.
phenylphosphonic acid ~10PPM relative to H3PO4
dimethyl methyl phoshate ~18 PPM
when I consider a standard, bandwidth, spectral resolution etc are a
concern for me. If they are not for you, then get DMMP, it is stable and
when used as an external standard it is fine in a sealed capillary. Also,
for quantification to get as good as you possibly can, make the standard
up in neat solute that the samples are in... i.e.- bacterial broth free of
phosphate (and autoclaved of course) then sealed... this should be fine.
... we have worried about susceptibility changes in the
ionic environment and as a way to ensure that lineshapes are similar (i.e.
comparing a narrow sharp standard to typical broad and fat in vivo signals
is a big mistake... read meyer, brown, nelson NMR biomedicine around 1988
or so on manual integration, the errors are spelled out nicely...
Internal triethyl phosphate is a possibility for some samples: 0.44 ppm
downfield of external 85% phosphoric acid. See JMR, 1986, 70, 484.
It depends on your machine. If you have short 90 degree pulses (i.e. large
excitation envelope) and a fast digitizer, I would recommend
dimethymethylphosphonate. It resonates at 38.06 ppm. You can look up my
reference, and that has reference to Phil Kuchels defining work in the area:
Barry JA., McGovern KA, Lien Y-H, Ashmore, B and Gillies RJ (1993) "Dimethyl
Methylphosphonate (DMMP): A 31P NMR Spectroscopic probe of intracellular volume in
mammalian cell cultures" Biochemistry 32,4665-4670
Pierre-Marie Robitaille and George Brown did some work on 31P
shifts (JMR 92, 73-74 (1991) and Comp Biochem Physiol Vol 87B
#2 pp 285-296 (1987)) They used methylenediphosphonic acid
in D2O (18.72ppm) and methyl phosphonate (11.95ppm).