qnmr (eBook)

1

FOREWORD

When I moved from university to an NMR laboratory in the chemical industry at the end of the 1980s, I wanted to apply my experience with quantitative NMR from my graduate studies to real laboratory operations. For this purpose, I had quantitatively evaluated some of the results of the normal NMR spectra and presented them to colleagues from other areas of analysis, mainly chromatography. A dear colleague replied to me as follows:

"An analyst would rather use his colleague's toothbrush than his method."

For more than 30 years I have been successfully applying quantitative NMR spectroscopy in a professional setting, but still have the feeling that not much has changed regarding the above statement. Understandably, there is still a distrust of qNMR on the part of mainstream methodologists from the HPLC laboratories and specialists in elemental analysis, but surprisingly also from within the ranks of NMR spectroscopy itself. Even the term “qNMR” is a pleonasm because NMR always has the potential to be interpreted quantitatively – only the precision is affected by the details of the experiment and analysis. NMR spectroscopists often take for granted important, distinguishing points about NMR that deserve restating: the method is non-destructive, and no physical separation of chemical species is required.

This book is intended to give everyone the opportunity to make friends with qNMR as a reliable method that offers the solution to a variety of problems in pharmaceutical analysis, food analysis, or even environmental analysis or diagnostics. For some of us – by which I mean pioneers and advocates of qNMR – the method clearly is most discussed in the metrology discipline, alongside primary methods such as weighing or coulometry. Through logical considerations and unambiguous experiments, all quantitative measurements can, in principle, be traced back to a uniform standard, e.g., water.

One does not have to solve all analytical quantitative problems using NMR spectroscopy, but a lot can be achieved. Let's turn this statement around: you don't have to solve everything with HPLC or titration, even if you have always done so. Technical and financial efforts must be considered to decide on the right method. In addition, there is always the need to consider the principle of traceability to SI, or to judge the robustness of the method under GxP.

This book is not a collection of methods, but it is intended to lay the foundations for a general acceptance of NMR in the canon of classical organic and inorganic analysis. It will hopefully be an eye-opener for many readers, as well as a blueprint for successful and confident adoption and use of NMR for myriad quantitation tasks.

In April 2023, my mentor, Prof. Dr. Stefan Berger, passed away. He was not only my teacher but also a role model and friend. His practical application of NMR spectroscopy in daily work is accessible at every NMR workstation, notably through his books "100 and 200 Experiments." In the early 80s, he introduced an automated NMR system with sample changer and multiple access points.

Allow me to share an anecdote from the mid-80s. At that time, a complete research group had not yet formed, as two points can only make a straight line. Nevertheless, there was a desire to give a presentation each week in the weekly seminar, alternating each week per person. Stefan Berger spoke about FT NMR and the Ernst equation. Some things are unforgettable, such as his prophecy regarding Richard Ernst: "He will receive the Nobel Prize for this someday!"

Finally, we discussed an unresolved issue concerning isotope effects, going back to the roots. The problem may now be resolved, perhaps only philosophically, but the commitment to continue pursuing such matters in his spirit remains.

In our last conversation, we discussed an unresolved issue related to isotope effects, going back to the roots. The problem may be merely philosophical and now resolved, but the commitment to pursue such matters in his spirit remains his legacy.

Prof. Dr. Bernd Diehl, Spectral Service, July 2023

1.1.AESTHETICS, SEMANTICS, AND HOLISTICS

To establish and apply an analytical method based on aesthetic criteria is not a scientific argument. In the case of NMR spectroscopy, however, we can point out such aspects; this could certainly have the effect that both already active NMR analysts and sceptics from the chromatography guild think outside the box. First, a few philosophical words about semiotics and the linguistic origins of the term “spectroscopy”.

The human cognitive apparatus is limited to our five senses. In fact, these senses are our analytical tools, both from a qualitative and quantitative perspective. Because of the biological and physical limitations of these, our primary analytical instruments, we have discovered or invented tools that extend our limited near vision to everything from the cosmic level to the atomic and below.

The space of the molecules or even of the atoms resist direct observation, and quantum mechanical phenomena are somewhat ‘ghostly’ to us. The term spectroscopy can be split into two parts, the Latin “spectrum” and the Greek “σκοπεῖν” (skopein). Spectrum means “image”, whilst skopein means “to look at”. Spectroscopy is, therefore, “image viewing”. In a more modern sense, the term spectrum is closely related to a rainbow, if not to sociological terms. A more detailed consideration brings us a little closer to the original because spectrum also means “appearance” or “spirit”. In English, “spectral” is still completely connected to this linguistic origin.

Spectroscopy enables our human senses to observe the ghostly world of atoms and molecules that is otherwise hidden from us. Thus, NMR spectroscopy in particular - as the name suggests - penetrates the (atomic) core of matter.

Quantum mechanical phenomena are very closely related to symmetry and asymmetry, and indeed this is directly reflected in NMR spectra. For some, an NMR spectrum is a scraggy mountain range, whilst for others it is a structure of the highest aesthetic.

1.2.METALANGUAGES

The specialist language, and steps taken with qNMR are:

In the first step of an NMR investigation, an order amongst disordered states is created by the applied magnetic field. The second step is to perturb this order by exciting it with electromagnetic energy. It is not this perturbation that is measured in an NMR experiment, but the FID, the energy emitted when the system returns to the undisturbed, resting state. It is somewhat analogous to ringing a bell; the measurement is made by the acoustically decaying noise that we can directly receive with our sense of hearing and that our mind interprets (even without Fourier transformation).

Firstly, the FID provides a holistic picture of what is being inspected. However, it is presented in a form that is not directly readable by humans - a foreign language, so to speak, that requires translation. This translation is a mathematical operation, the Fourier transform, which turns the time domain spectrum into the familiar, human-readable, frequency domain spectrum. If you look closely, you can still see a ghostly spark in the mathematical solution of a Fourier transformation in the real and imaginary spectra.

For non-specialists, an NMR spectrum is also like a foreign language. A further translation step is required to convert this spectrum, a seemingly random mixture of seemingly random lines, into one or more molecular formulas. With these molecular formulas, trained chemists can at least use their language and converse in it. For a person who is not trained in science, of course, chemistry must be translated into words again, even into different real languages. The path of knowledge thus leads one through a multitude of necessary translations from one metalanguage to a higher and different one. Finally, the information reaches the questioner at some point, namely “What is this?” and “How much is it?”

Along the translation chain, some of the original information is inevitably lost at each step. A critical loss of information occurs when the spectrum is translated into a molecular formula. Even die-hard NMR spectroscopists sometimes forget to pass on the quantitative information of an NMR spectrum or even, indeed, that it exists.

More information may be lost in this cascade of metalanguages that were originally part of a holistic NMR experiment. For example, information about molecular dynamics and other interactions of matter may not be described in a non-destructive, contactfree space-time experiment. An NMR spectroscopist observes nature by making it vibrate like a bell without striking it. He does not break matter down into its constituent parts to reconstruct the amount and type of matter from the fragments of a mass spectrum, or separate and isolate individual substances from complex mixtures of such via chromatography.

These fundamental differences must be known and understood because they are the cause of many of the misunderstandings between users of NMR spectroscopy and chromatography.

Quantitative NMR must therefore be validated according to its own unique principles and not simply by adopting standard chromatographic procedures. The canon of necessary validation steps between NMR spectroscopy and chromatography is crucially different. Conversely, one should not demand experiments that are nonsensical for NMR spectroscopy, either out of lack of...