NMR has won its reputation as a powerful tool for structure determination of organic molecules. In addition to the information provided by chemical shifts and coupling constants, the quantitative relationships existing between the peaks (or groups of peaks - multiplets) arising from the various nuclides in the sample has proven pivotal for the assignment and interpretation of NMR spectra.
Despite the fact that the concept of quantitative NMR (qNMR) has been coupled to NMR since the early 1950, shortly after the technique's inception, it seems as NMR, as an analytical tool for quantitative analysis was firstly mentioned in 1963 by Jungnickel and Forbes [Anal. Chem., 1963, 35 (8), pp 938–942] who determined the intramolecular proton ratios in 26 pure organic substances and Hollis [Anal. Chem., 1963, 35 (11), pp 1682–1684] who analyzed the amount fractions of aspirin, phenacetine and caffeine in respective mixtures.
From those pioneer works, many and varied studies on qNMR arose. As pointed out in J. Agric. Food Chem. 2002, 50, 3366-3374, qNMR is particularly suitable for the simultaneous determination of the percentage of active compounds and impurities in organic chemicals such as pharmaceuticals, agrochemicals and natural products, as well as vegetable oils, fuels and solvents, process monitoring, determination of enantiomeric excess, etc.
In what follows, I will use the term qNMR to refer to any quantitative measurement of NMR signals, regardless of whether the technique is employed as an analytical method (e.g. determination of the relative amounts of the components in a mixture) or as tool for structure determination or conformational analysis.
What’s the deal with qNMR?
The basic principle of qNMR assays is that, ideally, the integral of the set of all peaks which can be assigned to a particular nucleus is proportional to the molar concentration of that nucleus in the sample. Theoretically, this holds quite well, though there are deviations from the rule in strongly coupled systems. An important point to keep in mind is the word “ideally”; this includes, for example, perfectly relaxed samples.
Even so there remain a number of problems which can be first of all divided into two categories:
Sources of statistical assessment errors (scatter)
Sources of systematic assessment deviations (bias)
I will cover these points in detail in separate posts.
Intramolecular vs Intermolecular (mixtures) qNMR
The most important fundamental concept of qNMR is based on the fact that, the absorption coefficient for the absorption of electromagnetic energy is the same for all nuclides of the same species, regardless whether they belong to one or several molecules (e.g mixture). As a result, the NMR signal response (more precisely the integrated signal area) is directly proportional to the number of nuclides contributing to the signal.
For example, all organic chemists are very familiar with integrating the multiples of a 1H spectrum to elucidate or confirm a particular molecular structure (see figure below)
This application can be classified as Intramolecular qNMR. NOE spectra, where the intensity is related to the distance between spins and represents the main basis for NMR as a tool in structural molecular biology, is another application of Intramolecular qNMR (Note: In this context I’m not including Transfer-NOE used e.g. to study the structure of a ligand in a complex under conditions of fast exchange)
Let’s consider now another example, Intermolecular qNMR:
Purity determination of a compound using an internal standard (is) with known purity and assuming instrumental parameters properly set is given by the equation below (see for example, 10.1002/mrc.2464):
% purity by weight = W(is)/W(s) * A(s)/A(is)*MW(s)/MW(is)*H(is)/H(s)
where W(s) and W(is) are the weights of the sample and ISTD, A(s) and A(is) are the integrals (areas) of the sample and ISTD peaks, MW(s) and MW(is) are the molecular weights of the sample and ISTD, and H(s) and H(is) are the number of hydrogens represented by the integral for the sample and ISTD, respectively.
[NMR paper] Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monom
Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding.
Related Articles Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding.
J Mol Biol. 2002 Sep 6;322(1):137-52
Authors: Akerud T, Thulin E, Van Etten RL, Akke M
Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound...
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11-24-2010 08:58 PM
[NMR paper] NMR structure reveals intramolecular regulation mechanism for pheromone binding and r
NMR structure reveals intramolecular regulation mechanism for pheromone binding and release.
Related Articles NMR structure reveals intramolecular regulation mechanism for pheromone binding and release.
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14374-9
Authors: Horst R, Damberger F, Luginbühl P, Güntert P, Peng G, Nikonova L, Leal WS, Wüthrich K
Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the...
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[NMR paper] Molecular dynamics-derived conformation and intramolecular interaction analysis of th
Molecular dynamics-derived conformation and intramolecular interaction analysis of the N-acetyl-9-O-acetylneuraminic acid-containing ganglioside GD1a and NMR-based analysis of its binding to a human polyclonal immunoglobulin G fraction with selectivity for O-acetylated sialic acids.
Related Articles Molecular dynamics-derived conformation and intramolecular interaction analysis of the N-acetyl-9-O-acetylneuraminic acid-containing ganglioside GD1a and NMR-based analysis of its binding to a human polyclonal immunoglobulin G fraction with selectivity for O-acetylated sialic acids.
...
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08-22-2010 02:20 PM
[NMR analysis blog] Basis on qNMR: Integration Rudiments (Part II)
Basis on qNMR: Integration Rudiments (Part II)
My last post was a basic survey on different measurement strategies for peak areas. Manual methods such as counting squares or cutting and weighing, known as ‘boundary methods’ were introduced for historical reasons. These methods were first used by engineers, cartographers, etc, end then quickly adopted by spectroscopists and chromatographers.
In the digital era, most common peak area measurement involves the calculation of the running sum of all points within the peak(s) boundaries or by other quadrature method (e.g. Trapezoid, Simpson,...
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08-21-2010 09:12 PM
[NMR analysis blog] Basis on qNMR: Integration Rudiments (Part I)
Basis on qNMR: Integration Rudiments (Part I)
First a quick recap. In my last post I put forward the idea that integration of NMR peaks is the basis of quantitative analysis. Before going any further, I would like to mention that, alternatively, peak heights can also be used for quantitation, but unless some special pre-processing is employed (see for example P. A. Haysa, R. A. Thompson, Magn. Reson. Chem., 2009, 47, 819 – 824, doi) measurement of peak areas is generally the recommended method for qNMR assays.
In this post I will cover some very basic rudiments of NMR peak areas...
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08-21-2010 09:12 PM
[NMR analysis blog] Basis on qNMR: Rudiments
Basis on qNMR: Rudiments
http://3.bp.blogspot.com/_-MfflvAgRls/SwgqmEW0Y2I/AAAAAAAAAgs/kvnVoDo_Cms/s400/Intro1.jpgWhen I started playing drums, so many years ago, I kept hearing about so-called "Drum Rudiments". By that time, I was too young to realize how important they were and to me, they appear just as boring and repetitive exercises. However, rudiments (basic building blocks or "vocabulary" of drumming) are absolutely essential to master drums (something I have to admit I never achieved :-) )
In the last few years I’ve had the opportunity to meet and interact with many chemists...