Liquids Analysis Techniques

NMR Spectroscopy for Liquids

By far the most common use of NMR spectroscopy, studies of liquids, including samples dissolved in solvent, can achieve well-defined, high resolution data. NMR of solids, in contrast, must account for signal variations due to the orientation of molecules with respect to the baseline NMR magnetic field. In liquids, the rapid tumbling of molecules averages these anisotropic (direction-dependent) components to zero. As a result, solution-state NMR spectroscopy provides narrow spectral lines with clearly defined multiplets, revealing the fine detail of interactions among nuclei and electrons. (This effect is sometimes imitated in solid-state NMR by spinning the sample rapidly at a specific angle, called the magic angle.)

The Pulse Sequence

See NMR 101 for a general outline of a simple, one-dimensional liquid NMR experiment.

The timing, frequencies, and intensities of the RF pulses in an NMR experiment determine which nuclei are affected, and what aspects of their environment can be observed.

Important components revealed in liquid NMR experiments include:

Electron interactions:

As moving charged particles, nearby electrons affect the local magnetic field experienced by atomic nuelci. This appears in an NMR spectrum as a shift in resonance frequency, relative to a standard for that nucleus. Called chemical shift, these are small effects, expressed in parts per million. Smaller molecules, including many organics, can be identified by their pattern of chemical shifts in a 1H NMR spectrum.

Nuclear interactions:

Magnetic nuclei influence each other in a phenomenon called dipole coupling or spin–spin coupling. On an NMR spectrum, spin coupling splits single peaks into two or more sub-peaks. Direct coupling, through the interaction of the magnetic fields these nuclei generate, provides information about the distance between nuclei, and therefore molecular geometry. Indirect coupling, called J-coupling, happens through the effects of each magnetic nucleus on electrons in the molecule, which in turn influence the magnetic environment of other nuclei. J-coupling occurs strictly between nuclei linked through a small number of bonds, including hydrogen bonds. J-coupling between groups of protons in 1H NMR generate sets of multiple, close-spaced peaks called multiplets, that are observable only in liquid NMR thanks to its high resolution.

Each type of NMR experiment uses one or more characteristic pulse sequences to probe specific information about its target nuclei.

Hydrogen NMR

Providing high sensitivity and sharp signals, Proton (1H) NMR is the most common type of NMR experiment. Samples for 1H NMR must usually be dissolved in deuterated solvent to reduce background signal.

1H NMR spectrum for ethanol, showing spin coupling (green H signal split into sub-peaks). (Image by T.vanschaik (Own work) [GFDL or CC BY-SA 3.0], via Wikimedia Commons.)
1H NMR spectrum for ethanol, showing spin coupling (green H signal split into sub-peaks). (Image by T.vanschaik (Own work) [GFDL or CC BY-SA 3.0], via Wikimedia Commons.)

Carbon 13 and Other Elements

13C NMR is much less sensitive than 1H NMR, but it has a larger range of chemical shifts, making it more flexible for larger molecules where 1H peaks may overlap. A typical 13C experiment compares chemical shifts to those of expected moieties.

In principal, NMR can capture signals from any magnetic nucleus, including isotopes of almost every atomic element. Responses from heavier elements take longer – around eight seconds for the free-induction decay of 13C – so data acquisition with enough repetitions for good signal-to-noise can take minutes to hours.

13C NMR spectrum for ethanol, showing chemical shift (PPM) of carbon nuclei. (Image by Chris Evans [CC0], via Wikimedia Commons.)
13C NMR spectrum for ethanol, showing chemical shift (PPM) of carbon nuclei. (Image by Chris Evans [CC0], via Wikimedia Commons.)

Multi-dimensional NMR

For large molecules such as proteins and nucleic acids, single-acquisition spectra become crowded with overlapping signals from the many nuclei. Multidimensional NMR experiments deal with this issue by examining correlations in the results from two or more pulse sequences, each targeting either the same type of nucleus (homonuclear) – often 1H – or different ones (heteronuclear) – for example, 1H and 13C.

There are many variations on multi-dimensional NMR, taking advantage of different interactions among magnetic nuclei and their electronic environments. A typical 2D NMR experiment will run two, 1D pulse sequences in series with a suitable delay period between them, then analyze correlations between the two. Experiments with three and more dimensions may examine relationships among several species in order to place specific nuclei within a molecular structure.

Structural Studies

Multi-dimensional NMR experiments make it possible to determine 3D structures for large biomolecules, and to study structure-activity relationships. For example, triple-resonance NMR might be used to compare protein conformation before and after exposure to a drug candidate. In this scenario, pulse sequences targeting 1H, 13C, and 15N identify and establish relationships among specific atoms in the protein to reveal structure and conformation. Similarly, a suite of 1D, 2D and 3D NMR experiments can be run to determine the structure of a carbohydrate or nucleic acid.

Generally biomolecules are labeled with 13C and 15N for NMR study. Though naturally more abundant, 12C is not NMR-active, while the 14N nucleus presents a complex NMR response due to its quadrupole magnetic moment.

Dynamic NMR

NMR techniques to study chemical reactions over time vary with timescale. For slow reactions, on the order of seconds to minutes, NMR can be used to monitor changing concentrations of reaction products over time. Submillisecond processes average out over seconds-long NMR acquisition times, and chemical events happening over milliseconds to seconds will cause NMR line broadening. In these cases, reaction equilibria and rate constants can often be teased out by comparing NMR spectra as a function of temperature.