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Research

2D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)

Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does not have any diagonal peaks. All the cross-peaks indicate the protons directly attached to each carbon in a molecule. Similar to the COSY spectrum, imaginary lines are drawn in horizontal and vertical directions to identify the correlated peaks.
For example, the HSQC spectrum of 2-chlorobutane has a carbon signal at 11 ppm that can be assigned to the shielded methyl group and correlated to the proton multiplet at 1.01 ppm. The carbon signal at 24.8 ppm arises from the deshielded methyl group, which correlates to the proton multiplet at 1.52 ppm. Similarly, the methylene carbon signal at 33.3 ppm and methine carbon signal at 60.4 ppm correlate to the multiplets at 1.72 and 3.96 ppm, respectively.&#160; HSQC is a very useful technique for structurally elucidating complex molecules and is routinely used in protein NMR studies.

Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a ...

2D NMR: Overview of Heteronuclear Correlation Techniques

Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an X-nucleus, such as carbon-13 or nitrogen-15. This method is commonly used in nuclear magnetic resonance (NMR) spectroscopy to gain insights into complex chemical compounds&#39; structural and compositional aspects. A typical heteronuclear correlation spectrum displays X-nucleus chemical shifts on one axis and a proton spectrum on the other axis. Cross-peaks reveal the connection between specific protons and X-nuclei.
The main heteronuclear correlation experiments include HETCOR, HSQC, HMQC, and HMBC. HSQC, HMQC, and HMBC record the proton spectrum, whereas the X-nucleus spectrum is recorded by standard heteronuclear correlation spectroscopy or HETCOR. The HETCOR-HMQC-HSQC group of experiments cannot detect X-nuclei without an attached proton. HETCOR is used when ultrahigh peak resolution is required along the X-nucleus axis.
The heteronuclear single quantum correlation technique, or HSQC, investigates proton-to-X-nucleus single bond correlations. In an HSQC experiment, polarization is transferred from a proton nucleus to a neighboring X-nucleus and then back to the proton nucleus. The signal from the proton nuclei is then recorded. HSQC generates a spectrum like that of heteronuclear multiple-quantum correlation spectroscopy or HMQC but uses a different suppression method, providing better X-nucleus resolution along the axis.
Heteronuclear multiple-bond correlation, or HMBC, is a proton-detected experiment, like HMQC but with a longer initial delay time. This experiment is used to observe long-range proton to X-nucleus connectivity separated by 2-3 bonds, with some experiments going out to 4 or 5 bonds. In an HMBC experiment, direct one-bond correlations are suppressed as part of the sequence. The spectra of HMBC give rise to cross peaks that correlate which protons are coupled to what other specific X-nucleus is more than one bond away. The gradient HMBC (gHMBC) pulse sequence offers better suppression of interfering signals, making it particularly useful for analyzing complex molecules with overlapping signals.

Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an ...

2D NMR: Homonuclear Correlation Spectroscopy (COSY)

Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal and vicinal coupling are observed. For example, consider the COSY spectrum of ethyl acetate, where its 1D proton NMR spectrum is plotted along the vertical and horizontal axes with their corresponding chemical shift scale. Three spots on the diagonal corresponding to the three peaks in the 1D proton spectrum are called diagonal peaks. The COSY spectrum is symmetric about the diagonal, with signals above and below the diagonal called cross-peaks or off-diagonal peaks. An imaginary line is drawn from diagonal peaks parallel to the horizontal axis, leading to a cross peak. A vertical line is then drawn from the same cross peak towards the diagonal peak to correlate the peaks. The off-diagonal peaks correlating the multiplets at 1.25 and 4.2 ppm indicate that the methyl protons are coupled to the methylene protons. The signal at 2 ppm is not associated with cross-peaks because the protons are not involved in homonuclear coupling. A COSY spectrum is useful when overlapping multiplets complicate the corresponding 1D spectrum.

Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal ...

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and enhance the signals of low-abundance nuclei, such as carbon-13 and nitrogen-15, in small molecules. The fundamental principle behind INEPT is the transfer of polarization from a more abundant and highly polarizable nucleus, typically hydrogen-1, to the low-abundance nucleus of interest. This process effectively boosts the NMR signal of the low-abundance nuclei, making them more easily detectable.
The INEPT technique involves a series of carefully timed radiofrequency (RF) pulses applied to the sample. These RF pulses excite the more abundant nuclei, causing them to align their spins in a specific manner. Through the process of spin coupling, the polarization of the more abundant nuclei is then transferred to the low-abundance nuclei. As a result, the NMR signal of these low-abundance nuclei is significantly enhanced, allowing for a more detailed analysis of the molecular structure.
Various pulse sequences have been developed to improve the observability of these low-abundance nuclei when coupled to a high-receptivity nucleus. The INEPT pulse sequence combines a spin echo and selective population inversion, which includes 90&#176; and 180&#176; pulses on the high-sensitivity (the I spins) and low-sensitivity nuclei (the S spins), respectively. Coupling-constant-based delays between pulses facilitate magnetization transfer through space or bond interactions. Additional RF pulses and delays invert the low-gamma magnetization, producing an observable NMR signal. The net result is an enhanced NMR signal from low-gamma nuclei that would otherwise be challenging to detect.
Various pulse sequences have been developed to improve the detection of low-abundance nuclei when coupled with high-receptivity nuclei such as protons. The INEPT sequence yields enhanced signals for insensitive nuclei, with half of the CH group producing negative peaks and the other half positive peaks. This pattern allows for discrimination between different types of carbon-hydrogen bonds within the molecule.
Decoupling methods can be employed to enhance sensitivity and resolution in distinguishing various carbon-hydrogen bonds, making INEPT an invaluable tool in the chemical and pharmaceutical industries.

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and ...

Two-Dimensional (2D) NMR: Overview

The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse. The second step is the evolution or wait period t1, which is incremented systematically, and no data is observed during this period. The next step is the mixing period, in which a second radiofrequency pulse is introduced, which transfers the magnetization to the spins of nucleus B. In the last step, the magnetization of nuclear spins from B is detected during the acquisition time t2 for each t1. Time domains t1 and t2 are Fourier transformed into frequency domains f1 and f2, and these correspond to two dimensions of 2D spectra, while the third dimension is the intensity. In 2D experiments, coupling between the same kind of nucleus is called homonuclear 2D experiments, whereas coupling between two types of nucleus is called heteronuclear 2D experiments. 2D spectra can be displayed as stacked or contour plots. When presented as stacked plots, 2D spectra are difficult to interpret. The contour plot is the horizontal cross-section of a stacked plot where the size of the circles shows the signal intensity. Its interpretation is straightforward and it is preferred for data analysis.

The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily ...

2D NMR: Overview of Homonuclear Correlation Techniques

Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that utilizes a second 90-degree pulse to achieve high sensitivity and resolution. It excels in analyzing small to medium-sized molecules and provides information on directly coupled protons. COSY45, similar to COSY90, uses 45-degree pulses instead of 90-degree pulses. This results in reduced sensitivity but improved resolution.
Long-range COSY (LR-COSY) extends the COSY experiment to detect scalar couplings between nuclei further apart in the molecule. It achieves this by combining multiple pulse sequences and magnetization transfers.
Relayed COSY is a variant of COSY that adds extra pulses to the standard pulse sequence to produce cross-peaks between cross-peaks, showing specific molecular connections hidden by overlapping signals.
TOCSY (Total Correlation Spectroscopy), also known as HOHAHA (Homonuclear Hartmann-Hahn Spectroscopy), reveals all through-bond connectivity between all coupled nuclei. TOCSY is especially useful for analyzing molecules with overlapping signals and complex coupling patterns.
NOESY (Nuclear Overhauser Effect Spectroscopy) measures the nuclear overhauser effect, which arises from dipole-dipole interactions between nuclei. It provides information about spatial proximity and can be used to determine the three-dimensional structure of a molecule.
ROESY (Rotating-frame Overhauser Enhancement Spectroscopy) is similar to NOESY but employs a different mechanism to obtain through-space correlations. It provides information about spatial proximity and is particularly useful for studying molecules with weak NOE signals.
In conclusion, choosing a COSY variant depends on the molecule or reaction under investigation and the desired information. The right COSY variant can be determined based on molecular size, coupling complexity, and the need for spatial or long-range connectivity data.

Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations ...

Double Resonance Techniques: Overview

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or radiofrequency pulses to manipulate and observe two distinct nuclear spins. One important application of double resonance is spin decoupling, which selectively suppresses coupling with one type of nucleus while observing the NMR signal from another nucleus, simplifying the spectrum and enhancing resolution.
Spin decoupling is usually achieved by irradiating the sample atoms with a suitable radio frequency (rf) pulse sequence, effectively eliminating all the coupling with one nuclide. This simplifies the observed spectrum, making it easier to analyze and understand the relationships between different nuclei.
Double resonance experiments can be classified as heteronuclear or homonuclear, depending on whether the two sets of nuclei are of different isotopes or the same isotope, respectively. They can also be selective or nonselective, depending on whether the irradiating frequency covers only a portion or all of the resonance frequencies.
In nonselective heteronuclear decoupling, samples are exposed to an appropriate radiofrequency range to remove all coupling with one nuclide. However, increased external field strengths necessitate more powerful irradiation across broader frequency ranges. In certain instruments, continuous irradiation creates enough heat to cause damage to thermally sensitive samples.
To overcome these problems, modern methods using a series of pulses and exact timing delays, like J-modulated spin echo pulse sequences, can be employed to eliminate or adjust coupling effects within the spectrum.
For example, the attached proton transfer (APT) experiment applies a J-modulated spin echo pulse sequence to focus on the phase of detected carbon signals. Carbon atoms attached to an even number of protons exhibit positive signals in the spectrum, while those bonded to an odd number of protons appear as negative signals.
It utilizes a combination of a 180&#176; proton pulse and broadband decoupling to simplify spectral interpretation and assign multiplicities. The 180&#176; pulse is applied to the protons during the APT experiment. Its role is to refocus the spin coupling evolution that naturally occurs due to interactions between protons and carbons.
Specifically, after the 90&#176; pulse creates transverse magnetization, the 180&#176; pulse ensures that the effects of coupling are frozen during the initial period where the decoupler is turned off. This means the chemical shifts evolve only during the time 1/J&#8203; (the coupling constant period) after the first 180&#176; pulse.
By carefully timing this pulse sequence, the experiment isolates carbon multiplicities by utilizing the J-coupling between carbons and their attached protons. After the coupling evolution period (1/J&#8203;), broadband decoupling is turned on. This removes the J-coupling signals, collapsing multiplets into single peaks for each carbon resonance.
The decoupling ensures that carbon signals are free of splitting, allowing for a clear differentiation of signals based on phase (positive or negative) rather than splitting patterns.
The APT technique selectively enhances carbon signals, providing valuable information about carbon-proton connectivity, the number of attached hydrogen atoms, and the overall molecular structure.

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or ...

&sup1;H NMR of Labile Protons:  Deuterium (&sup2;H) Substitution

This lesson illustrates the role of deuterium substitution in simplifying the NMR spectrum of compounds comprising labile protons. One method employed is the use of deuterium. Amongst the three isotopes of hydrogen, deuterium (2H) has a nucleus composed of one proton and one neutron. When the D2O solvent is added to a pure dry ethanol solution, its labile proton is substituted with deuterium.
<img alt="Figure1" src="/files/ftp_upload/12280/12280-PT_Figure_1.svg" style="text-align: center; height: 700px;" />
Figure 1. The comparison of the proton NMR spectra of pure dry ethanol and the same in D2O solvent.
The signals from deuterium are not observed in a proton NMR spectrum, as deuterium&#8211;proton coupling is negligible, and no spin-spin splitting occurs between them. As shown in Figure 1, the NMR spectra of pure ethanol and ethanol in deuterium, the disappearance of the triplet in the latter, confirms that it corresponds to the labile &#8211;OH proton. Furthermore, the methylene protons that initially showed up as a multiplet transform to a quartet, proving that the multiplet is due to the coupling with the &#8211;OH proton and the methyl protons.

This lesson illustrates the role of deuterium substitution in simplifying the NMR spectrum of compounds comprising labile protons. One method employed is ...

Nuclear Overhauser Enhancement (NOE)

Irradiation of a spin-active nucleus causes an increase or decrease in the signal intensity of neighboring nuclei that are not necessarily chemically bonded or involved in J-coupling. &#160;This phenomenon, called the Nuclear Overhauser Enhancement (NOE), results from through-space interactions between the nuclear spins. The NOE effect decreases with increasing internuclear distance and is generally not observed beyond 4 angstroms. In NOE, dipole-dipole interactions between neighboring spin-active nuclei result in the transfer of nuclear spin polarization. These interactions can cause an increase or decrease in the signal intensity, which results in a positive or negative NOE, respectively.
NOE can be applied to improve the signal intensity of less sensitive nuclei such as carbon-13. Proton-decoupled carbon-13 spectra show a positive NOE effect, where the carbon signal intensities are substantially increased compared to those in proton-coupled spectra.
Targeted irradiation of specific nuclei can also reveal through-space proximity, which helps confirm stereochemistry and verify spectral peak assignments. The NOE effect is also applied in 2D NMR experiments, such as Nuclear Overhauser Effect Spectroscopy (NOESY), to determine the 3D structure of proteins.

Irradiation of a spin-active nucleus causes an increase or decrease in the signal intensity of neighboring nuclei that are not necessarily chemically ...

&sup1;H NMR of Labile Protons: Temporal Resolution

Protons bonded to heteroatoms such as nitrogen and oxygen exhibit a range of chemical shift values. This is due to the varying degree of hydrogen bonding between the proton and the heteroatom in other molecules. The extent of hydrogen bonding affects the electron density around the proton, thereby giving different chemical shift values for the protons in the proton NMR spectrum.
The &#8211;OH proton in alcohols typically appears in the range of &#948; 2 to 5 ppm but can vary depending on the specific alcohol and conditions. This &#8211;OH proton undergoes rapid proton exchange with other molecules and is referred to as a labile proton. So, the exact &#948; value of the &#8211;OH proton and the spin-spin coupling involving the &#8211;OH proton and the other protons in the alcohol depends on the rate of the &#8211;OH proton exchange. The proton exchange rate, in turn, depends on multiple factors, including the purity of the alcohol sample, temperature, and solvent.
<img alt="Figure1" src="/files/ftp_upload/12279/12279-PT_Figure_1.svg" style="text-align: center; height: 350px;" />
Figure 1. The triplet of a &#8211;OH proton in the proton NMR spectrum of neat ethanol.
For example, the &#8211;OH proton exchange in a pure dry ethanol sample is very slow on the NMR time scale, giving enough time for the spin-spin coupling. Consequently, as depicted in Figure 1, the proton NMR spectrum of pure dry ethanol shows a triplet for the &#8211;OH proton due to the spin-spin coupling with adjacent methylene protons. The methylene protons are seen as a multiplet due to the coupling with the &#8211;OH proton and the methyl protons.
The presence of trace amounts of an acid or base increases the proton exchange rate. The proton exchange becomes rapid, and the NMR spectrometer only records an average of all the possible environments for the &#8211;OH proton.
<img alt="Figure2" src="/files/ftp_upload/12279/12279-PT_Figure_2.svg" style="text-align: center; height: 325px;" />
Figure 2. The broad singlet of a &#8211;OH proton in the proton NMR spectrum of neat ethanol.
As a result, as recorded in Figure 2, the &#8211;OH proton exhibits a broad singlet. The protons from the adjacent methylene group present a quartet where the splitting is caused only by the adjoining methyl group.

Protons bonded to heteroatoms such as nitrogen and oxygen exhibit a range of chemical shift values. This is due to the varying degree of hydrogen bonding ...

&sup1;H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
<img alt="Figure1" src="/files/ftp_upload/13016/13016-PT_Figure_1.svg" style="text-align: center; height: 480px;" />
Figure 1. The temperature-dependent proton NMR spectra of cyclohexane.
Figure 1 depicts the proton NMR spectrum of the deuterium substituted sample, recorded at various temperatures. At room temperature, the rapid ring flipping of cyclohexane results in a single sharp peak. As the temperature is lowered to &#8722;60&#176;C, the rate of chair-chair interconversion reduces, resulting in the broadening of the peak.
Further lowering of the temperature broadens the peak, forming a saddle that splits into two peaks. On reaching a temperature lower than &#8722;89&#176;C, two sharp, well-resolved peaks corresponding to the equatorial and axial protons are formed. The peaks corresponding to the equatorial and axial protons occur at &#948; 1.62 and 1.14, respectively. At such low temperatures, the rate of chair-chair interconversion is reduced significantly, enabling the detection of the two distinct sets of protons on the NMR time scale.

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one ...

&sup1;H NMR of Conformationally Flexible Molecules: Temporal Resolution

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the axial and equatorial protons, resulting in a single peak at &#948; 1.4.
Similarly, NMR averages all the conformations of any molecule with rapid conformational equilibria. For example, the CH3 protons in bromoethane have a single resonance and coupling constant for splitting by the CH2 protons due to the rapid internal rotation of the carbon-carbon bond. Lastly, besides conformational equilibria, the time-averaging effect of NMR spectroscopy is also observed in some chemical reactions.

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately ...

Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation

The fragmentation patterns observed for compounds such as carboxylic acids, esters, and amides in the mass spectra include &#9082;-cleavage and McLafferty rearrangement. Fragmentation by &#9082;-cleavage preferentially occurs at the carbon-carbon bond at the &#9082;-position next to the carboxylic group to generate a neutral radical and a cation. Long chain compounds with hydrogen at their &#947;-carbon undergo McLafferty rearrangement to give a radical cation and a neutral alkene.
For example, the fragmentation of butyramide is demonstrated in Figure 1. The molecular ion peak of butyramide, which contains one nitrogen atom, shows an odd mass-to-charge ratio of 87. Fragmentation of butyramide molecular ion by &#9082;-cleavage produces a cation at a mass-to-charge ratio 44. In addition to &#9082;-cleavage, butyramide molecular ion also undergoes fragmentation via McLafferty rearrangement. The base peak at the mass-to-charge ratio 59 arises due to McLafferty rearrangement.
<img alt="Figure1" src="/files/ftp_upload/13051/13051_PT_Figure_1.svg" style="text-align: center; height: 250px;" />

The fragmentation patterns observed for compounds such as carboxylic acids, esters, and amides in the mass spectra include &#9082;-cleavage and McLafferty ...

Chemical Ionization (CI) Mass Spectrometry

The molecular ion peak of a molecule in the mass spectrum provides vital information for molecular identification. However, conventional electron impact ionization can lead to the rapid dissociation of some molecular ions before they reach the detector. A milder ionization method is required to increase the lifetime of such ionized analyte molecules. Chemical ionization (CI) is a gas-phase protonation reaction useful for mass-analyzing analyte molecules that are easily protonated to yield the corresponding conjugate acid. In this process, the sample is mixed with an excess reagent gas, which ensures the electron impact occurs primarily on the reagent gas. The charged species formed from the reagent protonates the analyte molecule, producing a relatively stable protonated analyte (conjugate acid) compared to the molecular ion. This results in an M+1 peak in the mass spectrum. The conjugate acid may then undergo fragmentation that generates additional signals.
For example, the CI of di-sec-butyl ether in methane gas illustrates this process. When the ether is mixed with excess methane gas as a reagent, electron impact occurs on methane rather than the ether. The resulting methane radical cation can react with another methane molecule to generate a methane radical and a methanium ion. The methanium ion is a source of gas-phase protons, which can protonate the ether to form its conjugate acid. The sequential reactions during the chemical ionization of di-sec-butyl ether are depicted in Figure 1.
<img alt="Figure1" src="/files/ftp_upload/12300/12300_PT_Figure_1.svg" style="text-align: center; height: 175px;" />
Figure 1: Chemical ionization of di-sec-butyl ether methane mixture.
This conjugate acid of the ether (m/z = 131) is relatively more stable than the molecular ion (m/z = 130) of ether formed via the conventional electron impact ionization. In the traditional route, the molecular ion undergoes fragmentation via &#945; cleavage, giving a signal at m/z = 101. Figure 2 illustrates the reactions that occur during electron impact ionization directly on di-sec-butyl ether.
<img alt="Figure2" src="/files/ftp_upload/12300/12300_PT_Figure_2.svg" style="text-align: center; height: 175px;" />
Figure 2: Electron impact ionization of di-sec-butyl ether and fragmentation of the molecular ion.
So, the mass spectra of di-sec-butyl ether ionized via CI feature an M+1 peak at m/z = 131. On the other hand, mass spectra of di-sec-butyl ether ionized via electron impact ionization do not show any peak at the m/z value of its molecular weight. Figures 3a and 3b show the mass spectra of di-sec-butyl ether ionized via the electron impact and chemical ionization methods, respectively.
<img alt="Figure3" src="/files/ftp_upload/12300/12300_PT_Figure_3.svg" style="text-align: center; height: 225px;" />
Figure 3:&#160;a) No m/z = 130 peak is visible in the mass spectrum obtained via electron impact ionization of di-sec-butyl ether. b) The m/z = 131 peak is clearly visible in the mass spectrum obtained via chemical ionization of di-sec-butyl ether.

The molecular ion peak of a molecule in the mass spectrum provides vital information for molecular identification. However, conventional electron impact ...

Mass Spectrometry: Alkyl Halide Fragmentation

Chlorine isotopes exist as 35Cl and 37Cl in a 3:1 ratio, while bromine isotopes exist as 79Br and 81Br in a 1:1 ratio. The mass spectrum of alkyl halides typically produces two distinct molecular ion peaks, the molecular ion peak, [M], and the molecular ion plus two, [M + 2] peak. The relative heights of these two peaks are proportional to the isotopic abundance ratios of the halide. For example, 2&#8208;chloropropane and 1&#8208;bromopropane display two peaks with relative peak heights in a 3:1 and 1:1 ratio, respectively.
Alkyl halides typically lose a non-bonding electron from the halogen to form molecular ions. The base peak results from the heterolytic cleavage of the carbon&#8211;halogen bond, where the bonding electrons go to the most electronegative atom, forming a cation and halogen atom. Interestingly, the &#9082;-cleavage is observed only in alkyl chlorides, not alkyl bromides. In alkyl chlorides, the&#160; C&#8211;Cl and C&#8211;C bonds exhibit similar strengths, while in alkyl bromides, the C&#8211;Br bond is weaker than the C&#8211;C bond. Consequently, the alkyl chlorides form a stable cation, where the positive charge is shared between the two atoms.
While less commonly discussed, fluoroalkanes and iodoalkanes exhibit unique fragmentation behavior. Fluoroalkanes, due to the strength of the C&#8211;F bond and the presence of only one isotope (19F), show limited fragmentation and lack isotopic peaks. Iodoalkanes, with their weak C&#8211;I bond and a single isotope (127I), predominantly fragment by losing iodine, resulting in simpler spectra with less isotopic splitting.

Chlorine isotopes exist as 35Cl and 37Cl in a 3:1 ratio, while bromine isotopes exist as 79Br and 81Br in a 1:1 ratio. The mass spectrum of alkyl halides ...

Electrospray Ionization (ESI) Mass Spectrometry

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the gaseous phase. The analyte biomolecule is premixed with an ionic liquid&#8212;a nonmolecular compound serving as the ionization source. The solution is sprayed through a high-voltage capillary to generate a fine aerosol. The voltage difference applied between the capillary and the entrance of the analysis chamber causes the movement of charged droplets toward the analysis chamber. The ionic liquid from the aerosol evaporates during this movement, leaving behind a variably protonated or ionized analyte molecule. These charged species are detected in the mass spectrum at an m/z value equivalent to the sum of the analyte molecule&#39;s molecular mass and the ions&#39; molecular weight from the ionic liquid associated with the analyte molecule.
The position of mass signals varies depending on the type of cations present in the ionic liquid. For example, the presence of sodium ions in the ionic liquid leads to an (M+23) peak in the mass spectra, while the presence of a proton results in an (M+1) peak.

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional ...

Matrix-Assisted Laser Desorption Ionization (MALDI)

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and characterization of various biomolecules, including proteins, peptides, nucleic acids, and carbohydrates. MALDI spectrometry is widely employed in biological and medical research, as well as in fields like pharmacology and biochemistry.
The analyte of interest, a biomolecule or a mixture of biomolecules, is mixed with a suitable matrix material. The matrix is usually an organic compound, such as a small organic acid or a crystalline substance, that facilitates ionization. This analyte-matrix mixture is then deposited onto a target surface, inserted into the mass spectrometer, and dried. During drying, the matrix forms small crystals that incorporate the analyte molecules within their structure.
Once the sample is prepared, a pulsed laser is directed onto the target surface. The matrix molecules absorb the laser beam energy, causing them to heat up and vaporize rapidly. This process is known as laser desorption. As the matrix molecules vaporize, they carry the embedded analyte molecules. The vaporized analyte molecules become ionized through a process called proton transfer. The proton transfer occurs between the matrix and the analyte molecule. The matrix molecule donates a proton to the analyte molecule, resulting in the formation of protonated ions. The analyte ions are then accelerated by an electric field and further separated based on their mass-to-charge ratio in a time-of-flight mass analyzer.
The mass spectrum obtained from MALDI spectrometry provides valuable information about the mass and relative abundance of the analyzed biomolecules.

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and ...

Mass Spectrometry: Aldehyde and Ketone Fragmentation

In mass spectrometry, the fragmentation of aliphatic aldehydes and ketones generally occurs through three key mechanisms: &#945;-cleavage, inductive cleavage, and the McLafferty rearrangement.
<ol>
	<li>&#945;-Cleavage 
	&#945;-Cleavage is a common fragmentation in carbonyl compounds where the bond adjacent to the carbonyl group is broken. This process produces an alkyl radical and an acylium cation, which is often detected in the mass spectrum due to its stability.</li>
	<li>Inductive Cleavage 
	In inductive cleavage, fragmentation yields an acyl radical and an alkyl cation. This occurs when electrons are drawn toward the carbonyl, weakening adjacent bonds and resulting in a distinctive fragmentation pattern. For example, in the mass spectrum of 5-methyl-2-hexanone, inductive cleavage produces an alkyl cation at a mass-to-charge (m/z) ratio of 71.</li>
	<li>McLafferty Rearrangement 
	The McLafferty rearrangement is a specific fragmentation pattern for carbonyl compounds with &#947;-hydrogen (hydrogen on the third carbon from the carbonyl group). The molecule undergoes a six-membered cyclic transition in this rearrangement, yielding a radical cation and a neutral alkene. For 5-methyl-2-hexanone, this rearrangement results in a peak at m/z 58.</li>
</ol>
Example: Fragmentation of 5-methyl-2-hexanone 
In the mass spectrum of 5-methyl-2-hexanone, the molecular ion undergoes these fragmentation patterns:
<ol style="list-style-type: circle;">
	<li>Inductive cleavage produces an alkyl cation at m/z 71.</li>
	<li>&#945;-Cleavage results in the base peak at m/z 43.</li>
	<li>McLafferty rearrangement creates a distinct peak at m/z 58.</li>
</ol>
An additional diagnostic feature in aldehydes is the M&#8722;1 peak, which arises due to the &#945;-cleavage of the aldehyde proton. In mass spectrometric analysis, this peak can help identify aldehydes, specifically among other carbonyl compounds.
Understanding these fragmentation pathways is crucial for interpreting mass spectra, particularly in identifying functional groups and distinguishing between similar compounds.

In mass spectrometry, the fragmentation of aliphatic aldehydes and ketones generally occurs through three key mechanisms: &#945;-cleavage, inductive ...

Mass Spectrometry: Alcohol Fragmentation

Alcohols (R-OH) ionize to lose one non-bonded electron from the oxygen atom, forming molecular ions. Due to their tendency to fragment rapidly, the intensity of the molecular ion peak in the mass spectrum is weak or sometimes absent. The fragmentation patterns for alcohols occur in two ways, i.e. &#9082;-cleavage and dehydration. During &#9082;-cleavage, the bond at the &#9082;-position adjacent to the hydroxyl group cleaves to give a resonance-stabilized cation and a radical. However, intramolecular dehydration involves losing a water molecule from an alcohol, forming an alkene cation, which shows a peak at M&#8722;18. Consider the fragmentation of butanol as shown below. &#9082;-cleavage produces a base peak at a mass-to-charge ratio of 31. In the case of dehydration, the fragmentation forms an alkene radical cation at a mass-to-charge ratio of 56.
<img alt="Figure1" src="/files/ftp_upload/13046/13046-PT_Figure_1.svg" style="text-align: center; height: 175px;" />

Alcohols (R-OH) ionize to lose one non-bonded electron from the oxygen atom, forming molecular ions. Due to their tendency to fragment rapidly, the ...

Mass Spectrometry: Alkyne Fragmentation

The fragmentation of alkynes preferentially occurs at the carbon&#8211;carbon bond between the &#945; and &#946; carbon of the alkyne bond to generate a 3-propynyl cation (or propargyl cation). In terminal alkynes, there is the only type of fragmentation that yields the 3-propynyl cation. The unsubstituted 3-propynyl cation exhibits a peak at a mass-to-charge ratio of 39. In internal alkynes, the 3-propynyl cation is substituted. For example, 2-pentyne fragments into methyl-substituted 3-propynyl cation, which subsequently features as a mass signal at the mass-to-charge ratio of 56. The 3-propynyl cation is resonance-stabilized, and the corresponding mass signal is intense in the mass spectrum of alkynes. Figure 1 shows the fragmentation of 1-pentyne into an ethyl radical and a resonance stabilized 3-propynyl cation.
<img alt="Figure1" src="/files/ftp_upload/13045/13045-PT_Figure_1.svg" style="text-align: center; height: 115px;" />
Figure 1: Fragmentation of 1-pentyne molecular ion to yield the 3-propynyl cation.
Another fragmentation pathway observed in terminal alkynes is the loss of hydrogen from the terminal sp carbon. This results in a mass signal with a mass-to-charge ratio of one unit less than the molecular ion. This M&#8211;1 peak is very intense and becomes the base peak in the mass spectra of many alkynes, so it can be used to identify alkynes.
<img alt="Figure2" src="/files/ftp_upload/13045/13045-PT_Figure_2.svg" style="text-align: center; height: 275px;" />
Figure 2: The mass spectrum of 1-pentyne.

The fragmentation of alkynes preferentially occurs at the carbon&#8211;carbon bond between the &#945; and &#946; carbon of the alkyne bond to generate a ...

Mass Spectrometry: Aromatic Compound Fragmentation

Upon ionization, aromatic compounds generate a molecular ion that is observed as a prominent peak in their mass spectra. For example, the molecular ion peak for benzene appears at a mass-to-charge ratio of 78, while toluene is observed at a mass-to-charge ratio of 92. The molecular ion benzene is highly stable and does not readily undergo further fragmentation due to the significant amount of energy required to disrupt the aromatic stability of the benzene ring. In contrast, the molecular ion of alkyl&#8208;substituted benzenes, such as toluene, can undergo fragmentation by losing a hydrogen atom, forming a benzylic carbocation. This carbocation can rearrange to form the resonance-stabilized tropylium cation, which appears in the mass spectra as a strong peak. In the case of toluene, this strong peak is observed at a mass-to-charge ratio of 91.

Upon ionization, aromatic compounds generate a molecular ion that is observed as a prominent peak in their mass spectra. For example, the molecular ion ...

Mass Spectrometry: Cycloalkene Fragmentation

The molecular ions of cycloalkenes undergo fragmentation via a retro-Diels&#8211;Alder reaction.

The reaction proceeds via the cleavage of two carbon-carbon bonds in the cycloalkene to yield ethene. The remaining part of the cycloalkene structure is a dienyl radical cation with a molecular weight of 28 u lower than the molecular ion. This fragmentation pathway is similar to the fragmentation of cycloalkanes releasing ethene, differing only in the resultant radical cation. An alkyl species forms from the cycloalkane, whereas a dienyl species originates from the cycloalkene.

Retro-Diels&#8211;Alder reactions of cycloalkenes are also similar to the McLafferty-rearrangement-type fragmentation observed in acyclic alkenes with hydrogen on the &#947; carbon. In both cases, cleavage occurs at two positions, yielding an ethene molecule and an alkenyl species. In acyclic alkenes, the cleavage occurs at one carbon-carbon bond and a carbon-hydrogen bond, along with rearranging hydrogen from one carbon to another. In cyclic alkenes, cleavage occurs at two carbon&#8211;carbon bonds without any atomic rearrangement. Figure 1 depicts the fragmentation of cyclohexene via a retro-Diels&#8211;Alder reaction.
<img alt="Figure1" src="/files/ftp_upload/13044/13044-PT_Figure_1.svg" style="text-align: center; height: 90px;" />
Figure 1: Fragmentation of cyclohexene via a retro-Diels&#8211;Alder reaction.
In addition to the retro-Diels&#8211;Alder reaction, branched cycloalkenes exhibit fragmentation via cleavage of the side chain to form a cycloalkene cation. For example, the fragmentation of 1-methyl-1-cyclohexene via a retro-Diels&#8211;Alder reaction and side-chain cleavage are shown in Figure 2.
<img alt="Figure2" src="/files/ftp_upload/13044/13044-PT_Figure_2.svg" style="text-align: center; height: 225px;" />
Figure 2: Fragmentation of 1-methyl-1-cyclohexene via (top) a retro-Diels&#8211;Alder reaction; (bottom) side-chain cleavage.
The corresponding signals of the isoprenyl radical cation and the cyclohexenyl cation are evident in the mass spectrum of 1-methyl-1-cyclohexene shown in Figure 3.
<img alt="Figure3" src="/files/ftp_upload/13044/13044-PT_Figure_3.svg" style="text-align: center; height: 250px;" />
Figure 3: Mass spectrum of 1-methyl-1-cyclohexene.

The molecular ions of cycloalkenes undergo fragmentation via a retro-Diels&#8211;Alder reaction.

The reaction proceeds via the cleavage of two ...

Mass Spectrometry: Amine Fragmentation

Amines can be identified using mass spectroscopy based on their characteristic fragmentation patterns. The molecular ions of amines undergo fragmentation via &#9082;-cleavage. The &#9082;-cleavage of the carbon-carbon bonds in amines generates an alkyl radical and resonance-stabilized nitrogen-containing cation.
In amines, the number of nitrogen atoms affects the mass of the molecular ion, which is described by the nitrogen rule of mass spectrometry. This rule states that a compound containing a single or an odd number of nitrogen atoms produces a molecular ion with an odd molecular weight, while a compound with no or an even number of nitrogen atoms generates a molecular ion with an even molecular weight.
For example, the mass spectrum of 3-methyl-1-butanamine, which contains one nitrogen atom, displays a molecular ion having an odd mass-to-charge ratio of 87. Fragmentation of this molecular ion via &#9082;-cleavage produces a base peak, which features a mass signal at a mass-to-charge ratio of 30, corresponding with the loss of the 2-methyl propyl radical.
Similarly, the fragmentation of triethylamine via &#9082;-cleavage generates a cation at a mass-to-charge ratio of 86. The molecular ion peak shows an odd mass-to-charge ratio of 101, as shown below.
<img alt="Figure1" src="/files/ftp_upload/13048/13048_PT_Figure_1.svg" style="text-align: center; height: 105px;" />

Amines can be identified using mass spectroscopy based on their characteristic fragmentation patterns. The molecular ions of amines undergo fragmentation ...

Mass Spectrometry: Long-Chain Alkane Fragmentation

The molecular ions of linear alkanes prefer to fragment at the carbon-carbon bond away from the end of the chain since the cleavage of an inner bond creates a stable carbocation and a stable radical. Consequently, the mass signals of linear alkanes feature intense peaks in the middle of the mass-to-charge ratio plot with weaker peaks on either end. The fragmentation of each carbon-carbon bond with the release of a methyl group in each splitting leads to prominent peaks in the mass spectra separated by 14 u. Smaller mass signals near each prominent peak result arise from secondary fragmentation at the carbon-hydrogen bond.
For example, consider the fragmentation pattern of n-hexane shown in Figure 1 and the corresponding mass spectra in Figure 2.
<img alt="Figure1" src="/files/ftp_upload/12299/12299-PT_Figure_1.svg" style="text-align: center; height: 500px;" />
Figure 1: Fragmentation of n-hexane molecular ion.
<img alt="Figure2" src="/files/ftp_upload/12299/12299-PT_Figure_2.svg" style="text-align: center; height: 275px;" />
Figure 2: Mass spectrum of n-hexane.
The signals at a mass-to-charge ratio of 71, 57, 43, 29, and 15 indicate that the cleavage can occur at all carbon-carbon bonds in the molecule. However, the relative abundance of these peaks varies. The difference between molecular ion signal and signal at 71 indicates the separation of methyl groups (M.W. = 15 u) group from molecular ions. Further release of a CH2 group from that fragment (M.W. = 14 u) results in a signal at 57. The signals at 43, 29, and 15 are due to the subsequent releases of CH2 groups. The abundance of signals at 15 and 71 is minimal due to the least probability for the formation of unstable methyl carbocation or a methyl radical, respectively. The base peak at 43 indicates the most preferred fragmentation, which yields a pair of stable carbocation and radical.

The molecular ions of linear alkanes prefer to fragment at the carbon-carbon bond away from the end of the chain since the cleavage of an inner bond ...

Cranial Nerves Exam I (I-VI)

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
During each section of the neurological testing, the examiner uses the powers of observation to assess the patient. In some cases, cranial nerve dysfunction is readily apparent: a patient might mention a characteristic chief complaint (such as loss of smell or diplopia), or a visually evident physical sign of cranial nerve involvement, such as in facial nerve palsy. However, in many cases a patient&#39;s history doesn&#39;t directly suggest cranial nerve pathologies, as some of them (such as sixth nerve palsy) may have subtle manifestations and can only be uncovered by a careful neurological exam. Importantly, a variety of pathological conditions that are associated with alterations in mental status (such as some neurodegenerative disorders or brain lesions) can also cause cranial nerve dysfunction; therefore, any abnormal findings during a mental status exam should prompt a careful and complete neurological exam.
The cranial nerve examination is applied neuroanatomy. The cranial nerves are symmetrical; therefore, while performing the examination, the examiner should compare each side to the other. A physician should approach the examination in a systematic fashion and go through the cranial nerves in their numerical order.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>I</td>
			<td>Olfactory</td>
			<td>Smell</td>
		</tr>
		<tr>
			<td>II</td>
			<td>Optic</td>
			<td>Visual acuity, afferent pupillary response</td>
		</tr>
		<tr>
			<td>III</td>
			<td>Oculomotor</td>
			<td>Horizontal eye movements (adduction), efferent pupillary response</td>
		</tr>
		<tr>
			<td>IV &#160;</td>
			<td>Trochlear</td>
			<td>Downward vertical eye movement, internal rotation of eye</td>
		</tr>
		<tr>
			<td>V &#160;&#160;</td>
			<td>Trigeminal&#160;&#160;&#160;</td>
			<td>Facial sensation, jaw movement</td>
		</tr>
		<tr>
			<td>VI &#160;</td>
			<td>Abducens</td>
			<td>Horizontal eye movement (abduction)</td>
		</tr>
		<tr>
			<td>VII</td>
			<td>Facial</td>
			<td>Facial movement and strength, taste, dampening of loud sounds, sensation; anterior wall of external ear canal</td>
		</tr>
		<tr>
			<td>VIII</td>
			<td>Acoustic</td>
			<td>Hearing, vestibular functioning</td>
		</tr>
		<tr>
			<td>IX</td>
			<td>Glossopharyngeal</td>
			<td>Movement of pharynx, sensation of pharynx, posterior tongue (including taste of posterior tongue), and most of ear canal</td>
		</tr>
		<tr>
			<td>X</td>
			<td>Vagal</td>
			<td>Movement and sensation of palate, pharynx, gag reflex, guttural sounds&#160;</td>
		</tr>
		<tr>
			<td>XI</td>
			<td>Spinal accessory</td>
			<td>Strength of sternocleidomastoids and trapezius muscles</td>
		</tr>
		<tr>
			<td>XII</td>
			<td>Hypoglossal</td>
			<td>Tongue protrusion and lateral movements</td>
		</tr>
	</tbody>
</table>
Table 1. The 12 Cranial Nerves and Their Basic Functions
Cranial nerve I (the olfactory nerve) is a purely sensory nerve that conveys the sense of smell, and is not routinely tested during most examinations. Cranial nerve II (the optic nerve) is the only cranial nerve that can be directly visualized as it exits from the central nervous system. Its axons convey visual information and compose the afferent limb of the pupillary reflex. Testing of the pupillary responses also assesses the function of cranial nerve III (the oculomotor nerve), parasympathetic fibers of which form the efferent limb of the pupillary reflex. Cranial nerve exam includes assessment of the extraocular movements, which are controlled by cranial nerves III, IV and VI. Cranial nerve III innervates the superior, medial, and inferior rectus muscles, as well as the inferior oblique muscle, which together function to move eyes medially and in the vertical plane. Cranial nerve IV (the trochlear nerve_ innervates the superior oblique muscles, which moves the eye downward and outward. Cranial nerve VI (the abducens nerve) innervates the lateral rectus muscles, which abducts the eyes. The function of the medial and lateral rectus muscles is straightforward: Lateral rectus is involved in abduction, meaning lateral movement along the horizontal plane. Medial rectus adducts moving the eye medially movement along the horizontal plane. The remaining muscles each causes movement in more than one direction and some combination of elevation/depression, abduction/adduction, intorsion/extorsion.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Muscle</td>
			<td>Innervation</td>
			<td>Primary action</td>
			<td>Secondary action</td>
			<td>Tertiary action</td>
		</tr>
		<tr>
			<td>Medial rectus</td>
			<td>CN III</td>
			<td>Adduction</td>
			<td>--</td>
			<td>--</td>
		</tr>
		<tr>
			<td>Superior rectus</td>
			<td>CN III</td>
			<td>Elevation</td>
			<td>Intortion</td>
			<td>Adduction</td>
		</tr>
		<tr>
			<td>Inferior rectus</td>
			<td>CN III</td>
			<td>Depression</td>
			<td>Extortion</td>
			<td>Adduction</td>
		</tr>
		<tr>
			<td>Inferior oblique</td>
			<td>CN III</td>
			<td>Extorsion</td>
			<td>Elevation</td>
			<td>Abduction</td>
		</tr>
		<tr>
			<td>Superior oblique</td>
			<td>CN IV</td>
			<td>Intorsion</td>
			<td>Depression</td>
			<td>Abduction</td>
		</tr>
		<tr>
			<td>Lateral rectus</td>
			<td>CN VI</td>
			<td>Abduction</td>
			<td>--</td>
			<td>--</td>
		</tr>
	</tbody>
</table>
Table 2. The function of the six extraocular muscles.
This first part of the cranial nerve exam concludes by testing cranial nerve V (the trigeminal nerve). This nerve has both motor and sensory components. It controls facial sensation, masticatory movements and forms the afferent limb of the corneal reflex. There are 3 major sensory branches of the trigeminal nerve - the ophthalmic, maxillary, and mandibular (also labeled V1, V2, and V3, respectively).

Anatomy and Physiology of the Cranial Nerves I -VI

Esame dei nervi cranici I (I-VI)

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
During ...

Cranial Nerves Exam II (VII-XII)

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
The cranial nerve examination follows the mental status evaluation in a neurological exam. However, the examination begins with observations made upon greeting the patient. For example, weakness of the facial muscles (which are innervated by cranial nerve VII) can be readily apparent during the first encounter with the patient. Cranial nerve VII (the facial nerve) also has sensory branches, which innervate the taste buds on the anterior two-thirds of the tongue and the medial aspect of the external auditory canal. Therefore, finding ipsilateral taste dysfunction in a patient with facial weakness confirms the involvement of cranial nerve VII. In addition, knowledge of the neuroanatomy helps the clinician to localize the level of the lesion: unilateral weakness of the lower facial muscles suggests a supranuclear lesion on the opposite side, while lesions involving the nuclear or infranuclear portion of the facial nerve manifest with an ipsilateral paralysis of all the facial muscles on the involved side. Cranial nerve VIII (the acoustic nerve) has two divisions: the hearing (cochlear) division and the vestibular division, which innervates the semicircular canals and plays an important role in maintaining balance. During a routine neurological examination, special testing of the vestibular nerve is usually not performed.
Cranial nerve IX (the glossopharyngeal nerve) and cranial nerve X (thevagus nerve) arise from the medulla and have laryngeal and pharyngeal function; their function is tested by assessing speech and motility of the soft palate.Because cranial nerves IX and Xform the sensory and motor limbs of the gag reflex, eliciting for gag reflex can also test their function. Cranial nerve XI (the spinal accessory nerve) innervates the sternocleidomastoid muscle and the upper portion of the trapezius muscle. These muscles control side-to-side turning the head and shrugging of the shoulders. The cranial nerve exam concludes by testing cranial nerve XII (the hypoglossal nerve), which provides motor control of the muscles of the tongue.
During the neurological assessment, the clinician should always be trying to tie together the findings of the exam to get insight into the underlying disease. The important diagnostic clues might include signs of multiple cranial nerve involvement and unilateral vs. bilateral cranial nerve dysfunction. It will help the clinician formulate differential diagnoses to know whether the patient&#39;s symptoms occurred suddenly (as expected with a stroke), over about a day (as in Bell&#39;s palsy), or gradually over weeks to months (as with an expanding mass lesion).
Evaluation of cranial nerves I-VI is covered in another video of this collection. This video demonstrates the systematic examination of cranial nerves VII-XII (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>I</td>
			<td>Olfactory</td>
			<td>Smell</td>
		</tr>
		<tr>
			<td>II</td>
			<td>Optic</td>
			<td>Visual acuity, afferent pupillary response</td>
		</tr>
		<tr>
			<td>III</td>
			<td>Oculomotor</td>
			<td>Horizontal eye movements (adduction), efferent pupillary response</td>
		</tr>
		<tr>
			<td>IV &#160;</td>
			<td>Trochlear</td>
			<td>Downward vertical eye movement, internal rotation of eye</td>
		</tr>
		<tr>
			<td>V &#160;&#160;</td>
			<td>Trigeminal&#160;&#160;&#160;</td>
			<td>Facial sensation, jaw movement</td>
		</tr>
		<tr>
			<td>VI &#160;</td>
			<td>Abducens</td>
			<td>Horizontal eye movement (abduction)</td>
		</tr>
		<tr>
			<td>VII</td>
			<td>Facial</td>
			<td>Facial movement and strength, taste, dampening of loud sounds, sensation; anterior wall of external ear canal</td>
		</tr>
		<tr>
			<td>VIII</td>
			<td>Acoustic</td>
			<td>Hearing, vestibular functioning</td>
		</tr>
		<tr>
			<td>IX</td>
			<td>Glossopharyngeal</td>
			<td>Movement of pharynx, sensation of pharynx, posterior tongue (including taste of posterior tongue), and most of ear canal</td>
		</tr>
		<tr>
			<td>X</td>
			<td>Vagal</td>
			<td>Movement and sensation of palate, pharynx, gag reflex, guttural sounds&#160;</td>
		</tr>
		<tr>
			<td>XI</td>
			<td>Spinal accessory</td>
			<td>Strength of sternocleidomastoid and trapezius muscles</td>
		</tr>
		<tr>
			<td>XII</td>
			<td>Hypoglossal</td>
			<td>Tongue protrusion and lateral movements</td>
		</tr>
	</tbody>
</table>
Table 1. The 12 cranial nerves and their basic functions

Anatomy and Physiology of the Cranial Nerves VII -XII

Esame dei nervi cranici II (VII-XII)

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
The ...

Motor Exam I

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
Abnormalities in the motor function are associated with a wide range of diseases, from movement disorders and myopathies to strokes. The motor assessment starts with observation of the patient. When the patient enters the examination area, the clinician observes the patient&#39;s ability to walk unassisted and the speed and coordination while moving. Taking the patient&#39;s history provides an additional opportunity to observe for evidence of tremors or other abnormal movements, such as chorea or tardive dyskinesia. Such simple but important observations can yield valuable clues to the diagnosis and help to focus the rest of the examination. The motor assessment continues in a systematic fashion, including inspection for muscle atrophy and abnormal movements, assessment of muscle tone, muscle strength testing, and finally the examination of the muscle reflexes and coordination. The careful systematic testing of the motor system and the integration of all the findings provide insight to the level at which the motor pathway is affected, and also help the clinician to formulate the differential diagnosis and determine the course of the subsequent evaluation and treatment.

Motor Exam I: Evaluation of Muscle Tone and Strength

Esame motorio I

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
...

Motor Exam II

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
There are two main types of reflexes that are tested on a neurological examination: stretch (or deep tendon reflexes) and superficial reflexes. A deep tendon reflex (DTR) results from the stimulation of a stretch-sensitive afferent from a neuromuscular spindle, which, via a single synapse, stimulates a motor nerve leading to a muscle contraction. DTRs are increased in chronic upper motor neuron lesions (lesions of the pyramidal tract) and decreased in lower motor neuron lesions and nerve and muscle disorders. There is a wide variation of responses and reflexes graded from 0 to 4+ (Table 1).
DTRs are commonly tested to help localize neurologic disorders. A common method of recording findings during the DTR examination is using a stick figure diagram. The DTR test can help distinguish upper and lower motor neuron problems, and can assist in localizing nerve root compression as well. Although the DTR of nearly any skeletal muscle could be tested, the reflexes that are routinely tested are: brachioradialis, biceps, triceps, patellar, and Achilles (Table 2).
Superficial reflexes are segmental reflex responses that result from stimulation of a specific sensory input (cutaneous or conjunctival) and the corresponding motor response. These reflexes include the corneal, conjunctival, abdominal, cremasteric, anal wink and plantar (Babinski) reflexes. The plantar reflex is a polysynaptic reflex elicited by stroking the lateral aspect of the sole with the normal response being plantar flexion of the great toe. This reflex changes with the normal development of the nervous system. In infants the toe will dorsiflex, but by 2 years of age the toe responds by plantar flexing. With damage to the pyramidal system, there is an unmasking of the more primitive reflex and the toe becomes &#34;upgoing&#34; or a positive Babinski sign.
The evaluations of coordination and gait are performed as a part of the neurological motor examination and can help a clinician localize lesions or recognize movement disorders. The coordination of movements and gait has complex multi-level regulation and requires an integrated function of different components of the nervous system. This part of the neurological examination allows the examiner to assess the function of the cerebellum, the cerebellar connections, and other tracts including brainstem structures. Coordination is assessed by looking for smooth and accurate movement, and requires the integration of sensory feedback with motor output, most of which occurs in the cerebellum. An impaired ability to coordinate the rate, range, timing, direction, or force of voluntary movement is called ataxia. Testing coordination includes evaluation of rapid alternating movements and point-to-point coordination, both of which can be altered as a result of cerebellar dysfunction. As in other parts of the examination, observation is the first step in the evaluation of the patient. A careful observation of a patient&#39;s gait can help the clinician screen for problems including weakness, movement disorders, spasticity, and cerebellar disease. No neurologic examination is complete without the assessment of gait. Occasionally, the only sign of a serious neurologic disorder is an impaired gait.

Motor Exam II: Reflex, Coordination, Gait and Station Testing

Esame motorio II

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
There ...

Sensory Exam

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
A complete sensory examination consists of testing primary sensory modalities as well as cortical sensory function. Primary sensory modalities include pain, temperature, light touch, vibration, and joint position sense. Sensation of the face is discussed in the videos Cranial Nerves Exam I and II, as are the special senses of smell, vision, taste, and hearing. The spinothalamic tract mediates pain and temperature information from skin to thalamus. The spinothalamic fibers decussate (cross over) 1-2 spinal nerve segments above the point of entry, then travel up to the brainstem until they synapse on various nuclei in thalamus. From the thalamus, information is then relayed to the cortical areas such as the postcentral gyrus (also known as the primary somatosensory cortex). Afferent fibers transmitting vibration and proprioception travel up to medulla in the ipsilateral posterior columns as fasciculus gracilis and fasciculus cuneatus, which carry information from the lower limbs and upper limbs, respectively. Subsequently, the afferent projections cross over and ascend to the thalamus, and from there to the primary somatosensory cortex.
The pattern of a sensory loss can help to localize the lesion and determine the diagnosis. For example, testing the primary modalities allows the examiner to distinguish between a length-dependent peripheral neuropathy (e.g., in diabetic patients), a radiculopathy from a possible cervical or lumbar herniated disc, or a dermatomal sensory level (e.g., in a spinal cord lesion).
In order to localize the sensory deficit, knowledge of neuroanatomy and the peripheral nervous system is crucial. When seeing a patient with a peripheral sensory deficit, it can be helpful to think about what nerve root(s) may be involved. A spinal nerve root arises from every spinal segment and consists of both a sensory dorsal root and a motor ventral root, which provide innervation to a specific dermatome and myotome, respectively. There are 31 paired spinal nerve roots: eight cervical, 12 thoracic, five lumbar, five sacral, and one coccygeal.
For example, the C5 through T1 roots form a network called the brachial plexus that controls movement and sensation in the upper limbs, including the shoulder, arm, forearm, and hand. The brachial plexus gives rise to the radial, median, and ulnar nerves. The median nerve carries sensation from all fingers except the fifth finger and half of the fourth, which are carried by the ulnar nerve. These nerve territories extend proximally on the palmar side of the hand. The ulnar and radial nerves carry sensory information from the dorsal side of the hands.
In the lower extremities, T12-L4 form the lumbar plexus, and L4-S4 form the sacral plexus. These plexi give rise to peripheral nerves. A few of these peripheral nerves are the femoral, obturator, and sciatic nerves (motor and sensory) and the lateral femoral cutaneous nerve (sensory only). The sciatic nerve gives rise to the tibial and common peroneal nerves. Use of a dermatomal and peripheral nerve map can be helpful in localizing sensory dysfunction in both the upper and lower extremities.
If primary sensory modalities are normal, cortical sensation (or higher order aspects of sensation) can be tested as well. Cortical sensation is tested when there is reason to suspect a disorder of the brain. Cortical sensory testing can assist with localization of nervous system disorders. The cortical sensory examination includes tests for tactile localization (extinction), stereognosis, graphesthesia, two-point discrimination, and point localization. Cortical sensory testing is not routinely done during a screening neurological examination.

Sensory Exam: Pathways and Peripheral Nerve Distribution

Esame neurologico

Source:Tracey A. Milligan, MD; Tamara B. Kaplan, MD; Neurology, Brigham and Women&#39;s/Massachusetts General Hospital, Boston, Massachusetts, USA
A ...

Neck Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
Examination of the neck can be a challenge because of the many bones, joints, and ligaments that make up the underlying cervical spine. The cervical spine is composed of seven vertebrae stacked in gentle C-shaped curve. The anterior part of each vertebra is made up of the thick bony body, which is linked to the body above and below by intervertebral discs. These discs help provide stability and shock absorption to the cervical spine. The posterior elements of the vertebra, which include the laminae, transverse, and spinous processes and the facet joints, form a protective canal for the cervical spinal cord and its nerve roots.
The cervical spine supports the head and protects the neural elements as they come from the brain and from the spinal cord. Therefore, injuries or disorders affecting the neck can also affect the underlying spinal cord and have potentially catastrophic consequences. The significant motion that occurs in the neck places the cervical spine at increased risk for injury and degenerative changes. The cervical spine is also a common source of radicular pain in the shoulder. For this reason, the neck should be evaluated as a routine part of every shoulder exam.

Neck: Palpation, Motion, Strength, Impingement, Neurologic Test

Esame obiettivo del collo

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
Examination of the neck can be a challenge because of the many bones, joints, ...

Shoulder Exam I

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
Examination of the shoulder can be complex, because it actually consists of four separate joints: are the glenohumeral (GH) joint, the acromioclavicular (AC) joint, the sternoclavicular joint, and the scapulothoracic joint. The GH joint is primarily responsible for shoulder motion and is the most mobile joint in the body. It has been likened to a golf ball sitting on a tee and is prone to instability. It is held in place by the four rotator cuff muscles (supraspinatus, infraspinatus, teres minor, and subscapularis), along with the GH ligaments.
The shoulder exam begins with the inspection and palpation of the key anatomic landmarks, followed by an assessment of the patient's range of motion. The opposite shoulder should be used as the standard to evaluate the injured shoulder, provided it has not been previously injured.

Shoulder Exam I: Palpation and Range of Motion

Esame obiettivo della spalla I

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
Examination of the shoulder can be complex, because it actually consists of four ...

Shoulder Exam II

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The shoulder exam continues by checking the strength of the rotator cuff muscles and biceps tendons. The rotator cuff muscles (supraspinatus, infraspinatus, teres minor, and subscapularis) act as compressors, holding the humeral head in place against the glenoid. Injury and degeneration of the rotator cuff tendons are the most common sources of shoulder pain. 
The strength testing of the rotator muscle is performed by testing motions against resistance applied by the examiner. Pain with these resisted motions suggests tendonitis; weakness suggests a rotator cuff tear. The strength tested is followed by tests for impingement syndrome, shoulder instability, and labrum injury. It is important to test both of the shoulders and compare between the sides. The opposite shoulder should be used as the standard to evaluate the injured shoulder, provided it has not been injured as well.

Shoulder Exam II: Muscle Strength, Impingement and Instability

Esame obiettivo della spalla II

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The shoulder exam continues by checking the strength of the rotator cuff muscles ...

Elbow Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The elbow is a hinged joint that involves the articulation of 3 bones: the humerus, radius, and ulna. It is a much more stable joint than the shoulder, and because of that, the elbow has less range of motion. The elbow and its structures are prone to significant injuries, particularly with repetitive motion. Lateral and medial epicondylitis (also called tennis elbow and golfer&#39;s elbow) are two common diagnoses and often occur as a result of occupational activities.
When examining the elbow, it is important to remove enough clothing so that the entire shoulder and elbow can be inspected. It is important to compare the injured elbow to the uninvolved side. A systematic evaluation of the elbow includes inspection, palpation, range of motion (ROM) testing, and special tests, including maneuvers to evaluate ligamentous stability and stretch tests to accentuate pain caused by epicondylitis.

Elbow Exam: Palpation, Stretch and Collateral Ligament Testing

Esame obiettivo del gomito

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The elbow is a hinged joint that involves the articulation of 3 bones: the ...

Wrist and Hand Examination

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The wrist is a complex joint made up of 8 carpal bones and their numerous articulations and ligaments. Overlying the wrist are the tendons and muscles of the hand and fingers. The hand is made up of 5 metacarpal bones, and the tendons that run to the hand overlie these bones. Finally, the fingers consist of 14 phalanges with their articulations held together by collateral ligaments and volar plates. Common mechanisms of both acute and chronic wrist injury include impact, weight bearing (which can occur in gymnastics), twisting, and throwing. Osteoarthritis of the hand commonly affects distal interphalangeal (DIP) and proximal interphalangeal (PIP) joints, while rheumatoid arthritis (RA) is seen in the metacarpophalangeal (MCP) and PIP joints.
It is important to compare the injured wrist or hand to the uninvolved side. Key aspects of the wrist and hand exam include inspection, palpation for tenderness or deformity, testing the range of motion (ROM) and strength, neurovascular assessment, ligaments and tendon testing, and the special tests.

Wrist and Hand: Motor, Sensory, Ligament and Tendon Testing

Esame del polso e della mano

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The wrist is a complex joint made up of 8 carpal bones and their numerous ...

Lower Back Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The back is the most common source of pain in the body. Examination of the back can be a challenge due to its numerous structures, including the bones, discs, ligaments, nerves, and muscles-all of which can generate pain. Sometimes, the location of the pain can be suggestive of etiology. The essential components of the lower back exam include inspection and palpation for signs of deformity and inflammation, evaluation of the range of motion (ROM) of the back, testing the strength of the muscles innervated by the nerves exiting in the lumbar-sacral spine, neurological evaluation, and special tests (including the Stork test and Patrick&#39;s test).

Lower Back Exam: Stork Test and Patrick&#039;s FABER Test

Esame della zona lombare

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The back is the most common source of pain in the body. Examination of the back ...

Hip Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The hip is a ball-and-socket joint that consists of the femoral head articulating with the acetabulum. When combined with the hip ligaments, the hip makes for a very strong and stable joint. But, despite this stability, the hip has considerable motion and is prone to degeneration with wear and tear over time and after injury. Hip pain can affect patients of all ages and can be associated with various intra- and extra-articular pathologies. Anatomic location of pain in the hip region can often provide initial diagnostic clues. Essential aspects of the hip exam include an inspection for asymmetry, swelling, and gait abnormalities; palpation for areas of tenderness; range of motion and strength testing; a neurological (sensory) exam; and additional special diagnostic maneuvers to narrow down the differential diagnosis.

Hip Exam: Trendelenburg, Hop, Leg Roll, Ober and FABER Tests

Esame obiettivo dell'anca

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The hip is a ball-and-socket joint that consists of the femoral head ...

Knee Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The knee is a hinged joint that connects the femur with the tibia. It is the largest joint in the body, and due to its location in the middle of the lower leg, it is subjected to a variety of traumatic and degenerative forces. Examination of the knee can be quite complex, owing to the fact it is an inherently unstable joint held together by various ligaments and supported by menisci, which act as shock absorbers and increase the contact area of the joint. In addition, the patella lies in front of the knee, acting as a fulcrum to allow the forceful extension of the knee needed for running and kicking. As the largest sesamoid bone in the body, the knee is a common source of pain related to trauma or overuse.
When examining the knee, it is important to remove enough clothing so that the entire thigh, knee, and lower leg are exposed. The exam begins with inspection and palpation of key anatomic landmarks, followed by an assessment of the patient's range of motion (ROM). The knee exam continues with tests for ligament or meniscus injury and special testing for patellofemoral dysfunction and dislocation of the patella. The opposite knee should be used as the standard to evaluate the injured knee, provided it has not been previously injured.

Knee Exam: Ligament, Meniscal and Patella Tests

Esame obiettivo del ginocchio

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The knee is a hinged joint that connects the femur with the tibia. It is the ...

Ankle Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The ankle and foot provide the foundation for the body and the stability needed for upright posture and ambulation. Because of its weight-bearing function, the ankle joint is a common site of injury among athletes and in the general population. Ankle injuries occur as a result of both acute trauma and repetitive overuse (such as running). The ankle is a fairly simple joint, consisting of the articulation between the distal tibia and talus of the foot, along with the fibula on the lateral side. The ankle is supported by numerous ligaments, most notably the deltoid ligament on the medial side, and laterally by three lateral ligaments: the anterior talofibular ligament (ATFL), the calcaneofibular ligament (CFL), and the posterior talofibular ligament (PTFL). 
Physical examination of the ankle and the patient history (including the mechanism of the injury and the location of pain) provide diagnostic information that helps the physician to pinpoint specific structures involved in an injury, and are essential for determining the severity of the injury and the subsequent diagnostic steps. When examining the ankle, it is important to closely compare the injured ankle to the uninvolved side. Essential components of the ankle exam include inspection, palpation, range of motion (ROM), and strength. In addition, special tests can assess the integrity of ligaments.

Ankle Exam: Talar tilt, Anterior drawer, Squeeze, Thompson Tests

Esame obiettivo della caviglia

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The ankle and foot provide the foundation for the body and the stability needed ...

Foot Exam

Source: <a href="https://www.jove.com/author/Robert+E._Sallis">Robert E. Sallis</a>, MD. Kaiser Permanente, Fontana, California, USA
The foot is a complex structure composed of numerous bones and articulations. It provides flexibility, is the essential contact point needed for ambulation, and is uniquely suited to absorb shock. Because the foot must support the weight of the entire body, it is prone to injury and pain. When examining the foot, it is important to remove shoes and socks on both sides, so that the entire foot can be inspected and compared. It is important to closely compare the injured or painful foot to the uninvolved side. The essential parts of the evaluation of the foot include inspection, palpation (which should include vascular assessment), testing of the range of motion (ROM) and strength, and the neurological evaluation.

Foot Exam: Range of Motion, Strength and Sensory Exam

Esame obiettivo del piede

Source: Robert E. Sallis, MD. Kaiser Permanente, Fontana, California, USA
The foot is a complex structure composed of numerous bones and articulations. It ...

Education Physical Examinations Iii

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Education Physical Examinations Iii

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Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

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Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)