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NMR Spectroscopy

how does NMR work

Core Concepts

In this tutorial, you will learn about NMR, or nuclear magnetic resonance. You will learn how it works and how chemists use this method to analyze compounds in organic chemistry!

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What is NMR?

NMR is a technique for analyzing structure and identifying compounds, often associated with organic chemistry. If you are conducting an experiment, you can use NMR to make sure you have the correct product, identify unknown products, or test purity. Organic chemists commonly use the technique for analyzing small organic molecules, while biochemists use this for complex protein identification.

How does it work?

The NMR instrument composition includes 3 different parts. First, there is a superconducting magnet that generates an external magnetic field around the sample. Second, a spectrometer transmits and receives radio waves. Last, a computer controls the instrument and performs data processing.

Broadly speaking, NMR extracts a spectrum from the magnetic properties of atomic nuclei to analyze and distinguish atomic nuclei. A sample of 2 – 50 mg of a product dissolved in a solvent is placed in the instrument’s magnetic field, which aligns the active nuclei. The energy transition from ground to excited state corresponds to radio frequencies, and when a nucleus returns to the ground (lower-energy) state, it emits radiation of the same frequency. The intramolecular magnetic field, specific to each molecule, can help give information about the structure and functional groups within the sample.

what is nmr

The signals that read off an NMR are called chemical shift peaks. The more electronegative a nucleus is, the higher the resonant frequency; furthermore, the resonant frequency of the energy jump depends on the chemical environment of each activated nucleus. Each active nucleus is seen as a peak in the NMR spectrum field. The location of each peak is dependent on multiple factors such as temperature, surrounding structures, and more; however, the most important one is the electronegativity/electropositivity. When a nucleus is electronegative (most electron withdrawing), it will have a lower chemical shift; on the other hand, when a nucleus is electropositive (most electron donating), it will have a higher chemical shift.

Physics of NMR

The fundamental physical concept behind NMR is the magnetic field generated by the spin of a nucleus. Each nucleus has a quantum number I, similar to the angular momentum quantum number of an electron. The number I can be a non-negative integer or half-integer (0, 1/2, 1, 3/2, . . . ). If nonzero, it generates a magnetic moment and corresponding magnetic quantum number mI, which takes values of I, I – 1, . . . –I. Through this magnetic moment, external magnetic fields and radio waves can interact with nuclei.

Left: alpha and beta spin states and Larmor precession in an external magnetic field B. Middle: the difference in energy between alpha and beta states created by the external field. Right: the basic NMR experiment in a schematic and a corresponding timing diagram.

In the NMR instrument’s magnetic field B, the magnetic moment of a nucleus aligns either with or against the field, respectively called the alpha and beta states (left). The magnetic moment (the arrow in the figure) rotates or “precesses” around the direction of the B field, at a characteristic frequency, the Larmor frequency. Interaction with the B field makes the alpha state lower in energy and the beta state higher in energy. As a result, a slight majority are in the alpha state before an experiment begins.

Pulse and Acquisition

In the most basic NMR experiment, a pulse of broad-band radio frequency (RF) radiation is directed at the sample. This shown as the black rectangle in the righthand diagram (diagrams like these depict “pulse sequences,” which can get very complex for specialized experiments). Photons with the exact Larmor frequency of the nuclei can be absorbed, flipping many of the nuclei from alpha to beta. After the pulse, the nuclei relax back to the lower-energy alpha state, releasing RF radiation in the process.

The collection of this emitted radiation is called free induction decay (FID) acquisition, because the intensity of the signal decays to zero. The example shown is for a single nucleus, and most FID curves are far messier. It takes Fourier transformation (a signal processing concept) to decompose the signal into individual frequencies and generate coherent spectra for interpretation.

Solvents

When creating a sample for NMR, you want to dissolve it in a solvent in order to give a good resolution result. If the sample must be analyzed as a solid, it requires a different machine, a magic angle spinning machine. The solvent should be deuterated, meaning it should have deuterium in the place of hydrogens. This is because other solvents containing hydrocarbons can have active nuclei, which will then show up on the spectra. By using these solvents, the chemical shifts may slightly shift, but the spectrum peak number will only represent the molecule you are analyzing. The most common solvent is deuterochloroform, CDCl3, although there are many other options such as DMSO and deuterated toluene and benzene.

Types of NMR

The two most common types of NMR are proton (1H) and carbon (13C); however, it can be applied to any sample which includes nuclei possessing spin.

1H NMR

1H (also called proton) NMR spectroscopy is specific to ordinary hydrogens, or protons. It is the most commonly used in the laboratory. 1H NMR can give you information on several topics:

  • Amount of unique hydrogens in the compound
  • Ratios of unique hydrogens
  • Chemical environment of each hydrogen
  • Surrounding hydrogens

13C NMR

C-13 spectroscopy is specific to the analysis of the carbons in a molecule. It is less common than H-1 but is still regularly used in organic chemistry laboratories. C-13 NMR can give the following information:

  • The number of unique carbons in the compound
  • Chemical environment of the compound

Other NMR Nuclides

In addition to 13C and 1H, there are an array of other nuclei (or nuclides) that are NMR active. For a nuclide to be NMR active, it must have a nonzero angular momentum quantum number (I). To be useful, it should also have a high enough natural abundance that some of the required nuclide will be present in a sample containing the element. If abundance is low, the technique is less sensitive (for example, 13C NMR is much less sensitive than 1H, because the abundance of the isotope 13C is only about 1%, while the abundance of 1H is nearly 100%).

Other nuclides commonly used for NMR are 15N, 19F, 31P, and 57Fe, which have the usual angular momentum quantum number I = 1/2. In addition to these, there are some with higher quantum numbers, such as:

  • 2H (deuterium), 14N and 6Li with I = 1
  • 11B, 23Na and 39K with I = 3/2
  • 17O and 25Mg with I = 5/2

Others such as 12C and 16O, although they may have high natural abundance, have I = 0, and are therefore undetectable using NMR.

Multidimensional NMR

Multidimensional NMR is a blanket term for various NMR techniques in which spectra are resolved over two or more axes instead of the usual one, giving additional structural information. These techniques are especially important for analysis of complex molecules such as proteins and other large biomolecules. Most structural information is not achievable from one-dimensional NMR spectra of these compounds.

Solid State NMR

This technique can also be performed on solids. However, as mentioned earlier, some special considerations are necessary when performing NMR on solid sample.

How to read NMR

There are numerous things you should look for when reading either a 13C or 1H NMR. Let’s go through them below for 1H-NMR:

Number of Peaks

The number of peaks corresponds to the number of unique hydrogens there are in the molecule. If there are 4 peaks, there are 4 unique protons. Remember that the number of peaks can but does not always represent the total number of hydrogens present in the molecule.

Additionally, we should keep in mind that there may be extra peaks that do not correspond to the molecule being analyzed. This could be for various reasons, such as contamination, impurities, reagents left over from a reaction, or simply the solvent. When using analyzing software, such as MestreNova, these peaks are often easy to identify and label. Usually they are not as large or tall as the peaks you are analyzing. Additionally, the location of the peaks is specific to the solvent, but you can check tables like these to see what impurities you might have.

Peak Shift

The X-axis on a an NMR spectrum represents the chemical shift in ppm (a unit of frequency independent of the field strength of the instrument). The shift in ppm decreases as you move left to right; higher values are referred to as downfield (left) while lower values are referred to as upfield (right).

As stated above, the main factor deciding the location of a peak shift is the electronegativity of a nucleus; but how do we know where to look for them?

The shift left or right on the spectrum can show information about the chemical environment. As a primary guide, protons that are a part of specific structures, such as functional groups, have a shift around where they are most commonly seen. Below is a table you can use when analyzing 1H NMR:

NMR shift table
Image from Brooks Cole Organic Chemistry Textbook

Keep in mind that different chemical environments cause peaks to shift one way or the other. For example, if the electron density surrounding a proton is high, there is ‘shielding,’ so the net magnetic field present at the nucleus is lower; greater shielding leads to resonance at lower frequencies, so the shift will be upfield to the right. On the other hand, if the electron density surrounding a proton is low, the nucleus is ‘deshielded,’ so it feels the external magnetic field more; greater deshielding leads to resonance at a higher frequency, so the shift will be downfield to the left.

Peak Integration

Integration is a process (automatic on modern NMR instruments) that calculates the area under each NMR peak, to indicate how many atoms the peak represents. The word integration comes from the calculus technique for finding the area under a curve. Peak integration does not directly provide the number of atoms, but it does give relative numbers.

Let’s take an example in 1H NMR. If peak A has integration 0.5 and peak B has integration 3, then the ratio of B to A protons is 6:1. In organic chemistry problems, integration information is often combined with the molecular formula of the compound, so that the exact number of atoms represented by each peak can be determined.

An approximate 1H spectrum of a simple molecule (ethyl chloride) showing integration and relative area. In this case, peak assignments (in blue) can be made from integration alone.

In MestreNova and other NMR analysis software, you can peak pick, or highlight it with a tool, in order to find the area under the peak to further see the comparison between peaks. Then you must normalize to the smallest significant peak and round to a whole number to get the number of hydrogens represented by each peak.

In rare cases, the peak heights can indicate the relative ratio of hydrogens in each peak. If there are two peaks and one is twice as high, it most likely means that the taller peak has twice as many unique hydrogens as the other peak. However, this is not reliable in most cases and peak integration is a much more accurate measure.

Splitting Pattern

There are various ways that each peak can split based on the structure of the molecule you are analyzing. If multiple hydrogens exist under a unique hydrogen peak, they could all have the same or slightly different chemical environments. For example, this could be because of the stereochemistry of the hydrogens at the same carbon are different; if one is on a dash and the other is on a wedge, they are at the same location but the atoms which they ‘see’ and interact with can be different because of the orientation. Because of this, the peak represented by these hydrogens can be broken into different splitting patterns.

Here are different splitting patterns that can be seen in throughout peaks:

nmr splitting patterns

Interpreting the Splitting Pattern

In general, the number of peaks corresponds to “n+1”, where n is the number of hydrogens within 3 bonds of each other, usually just on the adjacent carbons(s), within the same chemical environment. So if there is just one hydrogen on a carbon, that will correspond to 0+1 = 1, a singlet! If there is a unique hydrogen and on the carbon next door, there are 2 hydrogens attached, then that unique hydrogen is 2+1=3, a triplet!

When there are multiple adjacent carbons, you must take into account the chemical environment. Let’s say you have a perfectly symmetrical 3 carbon chain you want to analyze the peak splitting of the hydrogen of the middle carbon. On the first and third carbon there are 3 hydrogens on each. This corresponds to 6+1=7, a septet.

However, if they are not in the same chemical environment, they are not all counted as a group. When this happens, you follow a similar pattern rule: (n+1)(m+1). For example, let’s think about an asymmetric 3-carbon chain. If on the first carbon there is 1 hydrogen and 2 bromines and on the third hydrogen there are 2 hydrogens and a chlorine, then these two batches of hydrogen are not in the same chemical environment. Because of this, we have a (1+1)(2+1)= (2)(3)= 6, a doublet of triplets or a triplet of doublets!

How to use NMR Spectrum in Analysis (1H NMR Example)

We talked about how NMR is used to identify known or unknown compounds or test the purification of a product. Now that we know the details of NMR, let’s make more specific connections.

Below is an example of an NMR spectrum. This is a spectrum of a Biginelli reaction using methyl acetoacetate and 3,4,5-trimethyoxybenzaldehyde to create 3,4,-dihydropyrimidinone done by one of Chemtalk’s volunteers in Organic Chemistry laboratory.

1. Use spectra to work backwards

One way to use NMR is to analyze the peaks to find out the structure of a reaction product. After analyzing the spectra (here using MestreNova) you have information about the peaks, their shifts, the protons in each peak and you can even identify splitting patterns. Based on this, you can deduce which functional groups are present and the relative locations of the protons. It takes practice, but you can piece together many molecules simply from one spectrum.

2. Connecting the spectra to the reaction

If you have a reaction product you made, you can use the structure of the molecule to assign peaks. Chemical shift, integration, and splitting patterns all provide useful information for peak assignment.

3. Testing the purity

If you know the product you are trying to make, along with its structure, you can take idea 2 (above) and analyze the purity of your reaction scheme. If there are more peaks than unique hydrogens, there could still be reagents present or even some type of contamination.

A note on field strength and frequency of NMR instruments

Magnetic field strength limits the effectiveness of instruments, since this variable determines the resolution of spectra. Scientists often refer to instruments by the frequency which corresponds to their magnetic field strength. Early instruments were capable of about 100 MHz (megahertz), while current instruments generally range from about 300 MHz to 800 MHz (7-19 Tesla in field strength). The highest field-strength instrument built to date operates at 1.2 GHz or roughly 28 Tesla.

Further Reading

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