qNMR

Quantitative NMR (qNMR)

Principle:

The Quantitative NMR (qNMR) is based on one simple equation (1). The integrated intensity of a NMR peak is directly proportional to its molar concentration and to the number of nuclei that give rise to that resonance given that the NMR spectrum is obtained with care, especially long enough recycle delay. Quantitation is obtain without the need for calibration or corrections.

qnmr_0

For example the 1H NMR resonance of a methyl group would have 3 times the intensity of a peak resulting from a single proton.

A recent application (figure 1) by Kanatzidis group quantifying ammonium concentration in aqueous solution can be find here http://www.pnas.org/content/113/20/5530.short

Figure 1 The proton NMR spectra of N14NH4 ntensity of the peaks tells you how much it is. To validate this assay, a series of NH4Cl samples at different concentrations are made and 1H NMR spectra are collected. As you can see, the calculated NH4Cl concentrations are essentially same as the prepared ones.

Set up experiment:

Following points need to be addressed when setting up qNMR experiments:

  • Recycle delay (d1+at on VnmrJ): With equation Mt = Mmax(1-e-t/T1), one can calculated that with five times of T1, 99.3% of spins would be relaxed back to equilibrium. That’s sufficient for qNMR study.
  • Internal standard is usually picked based on following criteria:
    – chemically inert
    – low volatility
    – have similar solubility characteristics as the analyte
    – have reasonable T1 relaxation times
  • Digital resolution: The acquisition time (AT) is the time after the pulse for which the signal is detected. Because the FID is a decaying signal, there is not much point in acquiring the FID for longer than 3 x T2 because at that point 95% of the signal will have decayed away into noise. Typical acquisition times in 1H NMR experiments are 1 – 5 sec.
    DR = SW / NP, make sure to use long enough acquisition time to achieve high digital resolution.

When heteronuclear nuclei (i.e. 19F, 13C, and 31P etc.)  other  than proton are used to do qNMR, same precautions as proton need to be taken. Particularly when 13C is used, make sure NOE enhancement is turned off by using invgated pulse sequence.

Applications:

A large number of applications for qNMR are shown in below fugure, including Identity and purity assessment, quality control (QC) and quality assurance, overall characterization of foods and herbal products, as well as human metabolomic studies (figure 2). You can put many kinds of samples into a Universal NMR detector, and will get the quantitative information for different purposes. In addition, reaction monitoring, dynamics (measurement of activation energies), and diffusion measurements all rely on accurate quantitative measurements.

In all these applications, sizable errors can occur if procedures are not optimized or followed correctly. In “casual” measurements, error estimates are typically ±5%. Accuracies of < 0.5% can be achieved with more careful setup and analysis.

Figure 2 Sample varieties and panel of qNMR applications The current applications of qNMR can be divided in two main groups: (1) absolute quantitation and
purity determination of organic compounds (drugs, primary metabolites, natural products); and (2) metabolomics and quantitation of multiple analytes
in complex natural matrices (e.g., food, botanicals, biofluids). Essentially all types of metabolites (e.g., sugars, fatty acids, organic acids, steroids) can
be detected by NMR, explaining why a wide range of samples can be investigated. Therefore, qNMR applications cover the certification of purity, the
identification and quantitation of drug metabolites, the quality control of food products and herbal remedies, the identification of biomarkers in
complex natural matrices (e.g., herbal mixtures, biofluids), and finally clinical diagnosis (Guido Pauli etc. Current Option in Biotechnology, 2014).