Chapter Nuclear Magnetic Resonance (NMR) Spectroscopy direct observation of the H's and C's of a molecules. Nuclei are positively charged and spin on. NMR Spectroscopy. N.M.R. = Nuclear Magnetic Resonance. Basic Principles. Spectroscopic technique, thus relies on the interaction between material and. bulk magnetisation of an ensemble of spins will flip at a different angle with respect to the static field (B. 0.) ◇ After the pulse, each spin precesses individually.

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Nmr Spectroscopy Pdf

informative NMR parameters: chemical shift, spin-spin splitting, linewidths, requirements for NMR spectroscopy are rather stringent; the magnetic field should. The basic phenomenon of nuclear magnetic resonance NMR spectroscopy is NMR spectroscopy differs in a number of important aspects from other forms. LECTURE COURSE: NMR SPECTROSCOPY. 1. Table of Content. The physical basis of the NMR experiment. 5. The Bloch equations: 8. Quantum-mechanical.

Uses of NMR spectroscopy Nuclear Magnetic Resonance NMR spectroscopy is an analytical chemistry technique used in quality control and reserach for determining the content and purity of a sample as well as its molecular structure. For example, NMR can quantitatively analyze mixtures containing known compounds. For unknown compounds, NMR can either be used to match against spectral libraries or to infer the basic structure directly. Once the basic structure is known, NMR can be used to determine molecular conformation in solution as well as studying physical properties at the molecular level such as conformational exchange , phase changes, solubility, and diffusion. In order to achieve the desired results, a variety of NMR techniques are available. The basics of NMR are described here. If an external magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level generally a single energy gap. The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned. The basis of NMR Fig. Many nuclei such as deuterium 2H or hydrogen-2 have a higher spin and are therefore quadrupolar and although they yield NMR spectra their energy diagram and some of their properties are different. Chemical shift The precise resonant frequency of the energy transition is dependent on the effective magnetic field at the nucleus. This field is affected by electron shielding which is in turn dependent on the chemical environment. As a result, information about the nucleus' chemical environment can be derived from its resonant frequency. In general, the more electronegative the nucleus is, the higher the resonant frequency.

However, in some atoms such as 1H and 13C the nucleus does possess an overall spin. The rules for determining the net spin of a nucleus are as follows; If the number of neutrons and the number of protons are both even, then the nucleus has NO spin. If the number of neutrons plus the number of protons is odd, then the nucleus has a half-integer spin i. In the absence of an external magnetic field, these orientations are of equal energy. If a magnetic field is applied, then the energy levels split.

Each level is given a magnetic quantum number, m. When the nucleus is in a magnetic field, the initial populations of the energy levels are determined by thermodynamics, as described by the Boltzmann distribution. This is very important, and it means that the lower energy level will contain slightly more nuclei than the higher level. It is possible to excite these nuclei into the higher level with electromagnetic radiation.

The frequency of radiation needed is determined by the difference in energy between the energy levels. Calculating transition energy The nucleus has a positive charge and is spinning. This generates a small magnetic field.

The nucleus therefore possesses a magnetic moment, m, which is proportional to its spin,I. The constant, g, is called the magnetogyric ratioand is a fundamental nuclear constant which has a different value for every nucleus. The energy of a particular energy level is given by; Where B is the strength of the magnetic field at the nucleus. The difference in energy between levels the transition energy can be found from This means that if the magnetic field, B, is increased, so is DE.

It also means that if a nucleus has a relatively large magnetogyric ratio, then DE is correspondingly large. If you had trouble understanding this section, try reading the next bit The absorption of radiation by a nucleus in a magnetic field and then come back. The absorption of radiation by a nucleus in a magnetic field In this discussion, we will be taking a "classical" view of the behaviour of the nucleus - that is, the behaviour of a charged particle in a magnetic field.

This nucleus is in the lower energy level i. The nucleus is spinning on its axis. In the presence of a magnetic field, this axis of rotation will precess around the magnetic field; The frequency of precession is termed the Larmor frequency, which is identical to the transition frequency.

If energy is absorbed by the nucleus, then the angle of precession, q, will change. It is important to realise that only a small proportion of "target" nuclei are in the lower energy state and can absorb radiation.

There is the possibility that by exciting these nuclei, the populations of the higher and lower energy levels will become equal. If this occurs, then there will be no further absorption of radiation. The spin system is saturated. The possibility of saturation means that we must be aware of the relaxation processes which return nuclei to the lower energy state.

Relaxation processes How do nuclei in the higher energy state return to the lower state? Emission of radiation is insignificant because the probability of re-emission of photons varies with the cube of the frequency. NMR can be observed in magnetic fields less than a millitesla. Low-resolution NMR produces broader peaks which can easily overlap one another causing issues in resolving complex structures.

The use of higher strength magnetic fields result in clear resolution of the peaks and is the standard in industry. When placed in a magnetic field, NMR active nuclei such as 1 H or 13 C absorb electromagnetic radiation at a frequency characteristic of the isotope.

It is common to refer to a 21 T magnet as a MHz magnet since hydrogen is the most common nucleus detected, however different nuclei will resonate at different frequencies at this field strength in proportion to their nuclear magnetic moments.

An NMR spectrometer typically consists of a spinning sample-holder inside a very strong magnet, a radio-frequency emitter and a receiver with a probe an antenna assembly that goes inside the magnet to surround the sample, optionally gradient coils for diffusion measurements, and electronics to control the system.

Spinning the sample is usually necessary to average out diffusional motion, however some experiments call for a stationary sample when solution movement is an important variable. For instance, measurements of diffusion constants diffusion ordered spectroscopy or DOSY [5] [6] are done using a stationary sample with spinning off, and flow cells can be used for online analysis of process flows.

The vast majority of molecules in a solution are solvent molecules, and most regular solvents are hydrocarbons and so contain NMR-active protons.

The most widely used deuterated solvent is deuterochloroform CDCl 3 , although other solvents may be used depending on the solubility of a sample. The chemical shifts of a molecule will change slightly between solvents, and the solvent used will almost always be reported with chemical shifts.

NMR spectra are often calibrated against the known solvent residual proton peak instead of added tetramethylsilane. To detect the very small frequency shifts due to nuclear magnetic resonance, the applied magnetic field must be constant throughout the sample volume.

High resolution NMR spectrometers use shims to adjust the homogeneity of the magnetic field to parts per billion ppb in a volume of a few cubic centimeters. In order to detect and compensate for inhomogeneity and drift in the magnetic field, the spectrometer maintains a "lock" on the solvent deuterium frequency with a separate lock unit.

In modern NMR spectrometers shimming is adjusted automatically, though in some cases the operator has to optimize the shim parameters manually to obtain the best possible resolution.

It is a very weak signal, and requires sensitive radio receivers to pick up. A Fourier transform is carried out to extract the frequency-domain spectrum from the raw time-domain FID. A spectrum from a single FID has a low signal-to-noise ratio , but it improves readily with averaging of repeated acquisitions. Good 1 H NMR spectra can be acquired with 16 repeats, which takes only minutes. However, for elements heavier than hydrogen, the relaxation time is rather long, e.

Thus, acquisition of quantitative heavy-element spectra can be time-consuming, taking tens of minutes to hours. Following the pulse, the nuclei are, on average, excited to a certain angle vs. The extent of excitation can be controlled with the pulse width, typically ca. The pulse width can be determined by plotting the signed intensity as a function of pulse width.

Decay times of the excitation, typically measured in seconds, depend on the effectiveness of relaxation, which is faster for lighter nuclei and in solids, and slower for heavier nuclei and in solutions, and they can be very long in gases.

If the second excitation pulse is sent prematurely before the relaxation is complete, the average magnetization vector has not decayed to ground state, which affects the strength of the signal in an unpredictable manner. In practice, the peak areas are then not proportional to the stoichiometry; only the presence, but not the amount of functional groups is possible to discern.

An inversion recovery experiment can be done to determine the relaxation time and thus the required delay between pulses. A spinning charge generates a magnetic field that results in a magnetic moment proportional to the spin. Irradiation of the sample with energy corresponding to the exact spin state separation of a specific set of nuclei will cause excitation of those set of nuclei in the lower energy state to the higher energy state.

However, even if all protons have the same magnetic moments, they do not give resonant signals at the same frequency values.

This difference arises from the differing electronic environments of the nucleus of interest. Upon application of an external magnetic field, these electrons move in response to the field and generate local magnetic fields that oppose the much stronger applied field. This local field thus "shields" the proton from the applied magnetic field, which must therefore be increased in order to achieve resonance absorption of rf energy.

Such increments are very small, usually in parts per million ppm. For instance, the proton peak from an aldehyde is shifted ca. The difference between 2. However, given that the location of different NMR signals is dependent on the external magnetic field strength and the reference frequency, the signals are usually reported relative to a reference signal, usually that of TMS tetramethylsilane.

Additionally, since the distribution of NMR signals is field dependent, these frequencies are divided by the spectrometer frequency. However, since we are dividing Hz by MHz, the resulting number would be too small, and thus it is multiplied by a million.

This operation therefore gives a locator number called the "chemical shift" with units of parts per million. The chemical shift provides information about the structure of the molecule. The conversion of the raw data to this information is called assigning the spectrum. A typical CH 3 group has a shift around 1 ppm, a CH 2 attached to an OH has a shift of around 4 ppm and an OH has a shift anywhere from 2—6 ppm depending on the solvent used and the amount of hydrogen bonding.

While the O atom does draw electron density away from the attached H through their mutual sigma bond, the electron lone pairs on the O bathe the H in their shielding effect.

Fundamentals of Protein NMR Spectroscopy

In paramagnetic NMR spectroscopy , measurements are conducted on paramagnetic samples. The paramagnetism gives rise to very diverse chemical shifts. In 1H NMR spectroscopy, the chemical shift range can span ppm.

Because of molecular motion at room temperature, the three methyl protons average out during the NMR experiment which typically requires a few ms. These protons become degenerate and form a peak at the same chemical shift. The shape and area of peaks are indicators of chemical structure too. In the example above—the proton spectrum of ethanol—the CH 3 peak has three times the area of the OH peak. Software allows analysis of signal intensity of peaks, which under conditions of optimal relaxation, correlate with the number of protons of that type.

Analysis of signal intensity is done by integration —the mathematical process that calculates the area under a curve. The analyst must integrate the peak and not measure its height because the peaks also have width —and thus its size is dependent on its area not its height. However, it should be mentioned that the number of protons, or any other observed nucleus, is only proportional to the intensity, or the integral, of the NMR signal in the very simplest one-dimensional NMR experiments.

In more elaborate experiments, for instance, experiments typically used to obtain carbon NMR spectra, the integral of the signals depends on the relaxation rate of the nucleus, and its scalar and dipolar coupling constants. Very often these factors are poorly known - therefore, the integral of the NMR signal is very difficult to interpret in more complicated NMR experiments.

Some of the most useful information for structure determination in a one-dimensional NMR spectrum comes from J-coupling or scalar coupling a special case of spin-spin coupling between NMR active nuclei. This coupling arises from the interaction of different spin states through the chemical bonds of a molecule and results in the splitting of NMR signals.

For a proton, the local magnetic field is slightly different depending on whether an adjacent nucleus points towards or against the spectrometer magnetic field, which gives rise to two signals per proton instead of one. These splitting patterns can be complex or simple and, likewise, can be straightforwardly interpretable or deceptive.

This coupling provides detailed insight into the connectivity of atoms in a molecule. Coupling to additional spins will lead to further splittings of each component of the multiplet e. Note that coupling between nuclei that are chemically equivalent that is, have the same chemical shift has no effect on the NMR spectra and couplings between nuclei that are distant usually more than 3 bonds apart for protons in flexible molecules are usually too small to cause observable splittings.

Nuclear magnetic resonance

Long-range couplings over more than three bonds can often be observed in cyclic and aromatic compounds, leading to more complex splitting patterns. Related Information. Email or Customer ID. Forgot password? Old Password.

Nuclear magnetic resonance spectroscopy - Wikipedia

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