behavior

JoVE Davranış, insan ve/veya hayvan davranışlarını anlamak için gözlemsel ve deneysel teknikler sunar. Bu bölüm, genetik nedenlerin ve internal ve eksternal uyaranlara karşı fizyolojik tepkilerin analizinin yanı sıra ilgili beyin bölgelerini incelemek için yöntemler içermektedir.

Davranış

biochemistry

JoVE Biyokimya, biyomoleküllerin (proteinler, nükleik asitler, karbonhidratlar, lipitler ve metabolitler gibi) yapısı ve işlevi yanısıra, bu moleküllerin biyolojik süreçler sırasında etkileşimlerini ve dönüşümlerini derinlemesine anlamak için yöntemler sunmaktadır.

Biyokimya

bioengineering

JoVE Biyomühendislik, temel biyolojik süreçleri ve fonksiyonları anlamak üzere fizik ve yaşam bilimlerini birleştiren yöntem ve teknikleri kapsamaktadır. Bu bölüm aynı zamanda hastalıkların ölçüm, tanı ve tedavi süreçlerini iyileştirmek için fizik bilim araçlarını ve yaklaşımlarını da içermektedir.

Biyomühendislik

biology

JoVE Biyoloji, tüm genel biyoloji araştırma metodolojilerini memnuniyetle karşılar. Bu bölümün içeriği, standart tekniklerin yeni uygulamalarından yaşamın ve canlı organizmaların işlevlerini anlamayı amaçlayan yeni yaklaşımlara kadar hücre, moleküler ve organizma biyolojisinin tüm alanlarını kapsamaktadır. Bu çeşitli bölüm, fiziksel biyoloji, hücresel biyokimya, genetik, fizyoloji, sistem biyolojisi ve ökaryotik ve prokaryotik model sistemlerinin bir kombinasyonundaki teknikleri içerir, ancak bunlarla sınırlı değildir.

Biyoloji

cancer research

JoVE Kanser Araştırması, kanserin anlaşılmasını, tespit edilmesini, tedavi edilmesini ve önlenmesini amaçlayan biyomedikal araştırma ve klinik uygulamalardaki deneysel yaklaşımları göstermektedir. Bu bölümdeki video makaleleri, kanserin hücresel ve moleküler mekanizmalarını incelemeye yönelik yöntemleri, kanser araştırmalarında hayvan modellerini, yeni terapi ve teşhislerin geliştirilmesini, tümör görüntülemeyi, kimyasal ve radyasyon tedavilerini, cerrahi prosedürleri ve tedavideki gelişmeleri göstermektedir. Bu video makaleleri araştırmacılara ve klinik uygulayıcılara kanser araştırmalarındaki teknolojileri ve deneysel yöntemleri öğrenmenin etkili bir yolunu sunuyor.

Kanser Araştırmaları

chemistry

JoVE Kimya, analitik, organik, polimer, fiziksel ve inorganik kimya dahil olmak üzere çok çeşitli disiplinlerden yöntemler içermektedir. Bu bölüm aynı zamanda gelişmiş veya benzersiz uygulamalar için materyallerin ve küçük moleküllerin tasarım ve hazırlanması konularını kapsamaktadır.

Kimya

developmental biology

JoVE Gelişim Biyolojisi, biyolojik gelişmeyi in vitro ve/veya in vivo şekilde, moleküler, hücresel, doku, organ ve tüm organizma seviyelerinde incelemek için kullanılan yöntem ve teknikleri içermektedir.

Gelişim Biyolojisi

engineering

JoVE Mühendislik, fizik araştırmalarında kullanılan çok çeşitli deneysel ve enstrümantal teknikleri kapsar. Bu alandaki araştırmalar, fiziksel araçları kullanarak cihaz mekanizmaları ve verimlilikleri gibi çok çeşitli bilimsel soruları ele almaya ve yanıtlamaya çalışmaktadır. Bu yaklaşım genellikle uzmanlıkların bir kombinasyonunu gerektirir ve bu alandaki araştırmalar, makine, elektrik ve kimya mühendislerinin katkılarıyla disiplinler arası olma eğilimindedir.

Mühendislik

environment

JoVE Çevre Bilimi, çevresel kaygıları ele alan ve Dünya'nın ekosistemini daha iyi anlamaya çalışan araştırma metodolojilerine kendini adamıştır. Toplumun çevre üzerindeki etkisini değerlendiren, Dünya'nın kaynaklarını korumaya yönelik çözümler öneren ve sürdürülebilir yakıt kaynakları geliştiren deneysel metodolojilere özel önem verilmektedir. Çevreye duyarlı bir gezegene doğru ilerlemek, hem yaşam hem de fizik bilimcilerinin katkılarıyla ortak bir çabadır.

Çevre

genetics

JoVE Genetik, gen fonksiyonu ve düzenlemesini araştırmaya yönelik deneysel yaklaşımları gösteren video makaleleri yayınlamaktadır. Bu, gen ifadesini, mutajenezi, epigenetiği, gelişimin genetik belirleyicilerini, genetik bozuklukları, popülasyon genetiğini ve evrimi incelemeye yönelik yöntemleri içerir.

Genetik

immunology and infection

JoVE İmmünoloji ve Enfeksiyon, küresel sağlığı iyileştirmeye yönelik ortak bir girişime yönelik çalışan çeşitli alanlardan içerik içerir. Patojenlere verilen biyolojik tepkiyi moleküler düzeyden organizma düzeyine kadar değerlendiren tekniklerin yanı sıra terapötik ajanlar ve bunların hastalık tedavisindeki etkinlikleri de sunulmaktadır.

İmmünoloji ve Enfeksiyon

medicine

JoVE Tıp, temel araştırma ve klinik uygulamalar arasında bir bağlantı görevi görür. Makaleler tıbbi prosedürleri, vaka çalışmalarını veya klinik araştırma metodolojilerini sunabilir. Dahil edilen çalışmalar genellikle hastalığın tedavisine yönelik öngörücü, önleyici, kişiselleştirilmiş ve pratik yaklaşımlara yönelik olarak modellenmiştir. Bu bölümdeki araştırma metodolojilerinin amacı genel hasta bakımını ve prognozu iyileştirmektir.

Tıp

neuroscience

JoVE Nörobilim, beyin ve sinir sisteminin yapısını, fonksiyonunu, fizyolojisini ve patofizyolojisini araştırma yöntemlerini kapsamaktadır. Bu bölümde nörolojik hastalıklar ve bozukluklar için potansiyel tedavi platformları ve cerrahi tekniklere de yer verilmiştir.

Nörobilim

jove journal

JoVE Journal

Bilimsel video dergisi. Hakem değerlendirmeli. Çok disiplinli. PubMed ve Web of Science'da indekslenmiştir.

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Research

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: 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 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 ...

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 ...

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 ...

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 ...

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:  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 ...

&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 ...

&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 ...

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 ...

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: 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 ...

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 ...

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: 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 ...

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: 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: 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: 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: 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 ...

Basic Life Support:  Cardiopulmonary Resuscitation and Defibrillation

Source: <a href="https://www.jove.com/author/Julianna_Jung">Julianna Jung</a>, MD, FACEP, Associate Professor of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
High-quality cardiopulmonary resuscitation (CPR) is the single most important determinant of intact survival in cardiac arrest, and it is critical that all healthcare workers are able to perform this lifesaving technique effectively. Despite the conceptual simplicity of CPR, the reality is that many providers perform it incorrectly, resulting in suboptimal survival outcomes for their patients. This video looks at the essential elements of high-quality CPR, discusses the physiologic basis for each step, and describes how to optimize them in order to enhance survival outcomes. Appropriate prioritization of interventions in cardiac arrest and methods for optimizing resuscitation performance are covered as well.

Basic Life Support I: Cardiopulmonary Resuscitation and Defibrillation

Source: Julianna Jung, MD, FACEP, Associate Professor of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
High-quality ...

Basic Life Support Part II:  Airway/Breathing and Continued Cardiopulmonary Resuscitation

Source: <a href="https://www.jove.com/author/Julianna_Jung">Julianna Jung</a>, MD, FACEP, Associate Professor of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
High-quality cardiopulmonary resuscitation (CPR) and defibrillation are the most important interventions for patients with cardiac arrest, and should be the first steps that rescuers perform. This is reflected in the American Heart Association's new "CAB" mnemonic. While rescuers were once taught the "ABCs" of cardiac arrest, they now learn "CAB" - circulation first, followed by airway and breathing. Only once CPR is underway (and defibrillation has been performed, if a defibrillator is available) do we consider providing respiratory support. This video will describe the correct technique for providing respiratory support to a patient in cardiac arrest, and how to continue basic life support over the period of time until help arrives.
This video assumes that all the steps described in "Basic Life Support Part I: Cardiopulmonary Resuscitation and Defibrillation" have already been completed. This video does NOT depict the initial steps taken when arriving at the scene of a cardiac arrest.

Basic Life Support II: Airway/Breathing &amp; Cardiopulmonary Resuscitation

Pericardiocentesis

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA 
The heart lies within the pericardium, a relatively inelastic fibrous sac. The pericardium has some compliance to stretch when fluid is slowly introduced into the pericardial space. However, rapid accumulation overwhelms pericardial ability to accommodate extra fluid. Once a critical volume is reached, intrapericardial pressure increases dramatically, compressing the right ventricle and eventually impeding the volume that enters the left ventricle. When these chambers cannot fill in diastole, stroke volume and cardiac output are diminished, leading to cardiac tamponade, a life-threatening compression of the cardiac chambers by a pericardial effusion. Unless the pressure is relieved by aspiration of pericardial fluid (pericardiocentesis), cardiac arrest is imminent. 
Cardiac tamponadeis a critical emergency that can carry high morbidity and mortality. Patients may present in extremis, without much time to make the diagnosis and perform life-saving treatments. Causes of this condition are broken into traumatic and non-traumatic categories, with different treatment algorithms. Stab and gunshot wounds are the primary cause of traumatic tamponade, but it may occur from blunt trauma associated with sternal or rib fractures as well as shearing of vessels from rapid deceleration injuries. Non-traumatic causes include rupture of the aortic base from ascending aortic dissection, myocardial rupture of the ventricle following myocardial infarction, spontaneous bleeding from thrombolytic or anticoagulant medication, and effusions created by infection or cancer. 
Slowly growing chronic effusions are usually not life-threatening, even large ones. The pericardium has gradually stretched to incorporate liters of fluid in some cases. These may be treated with elective pericardiocentesis under fluoroscopic guidance, or a pericardial window. However, transition into tamponade physiology with a patient in extremis requires urgent pericardiocentesis, even with a small amount of fluid. Tamponade presents a challenge to diagnose, as its symptoms and physical findings are often nonspecific, common to a number of diseases. An electrocardiogram (ECG or EKG) may show electrical alternans, and a chest xray may show an enlarged "water bottle" cardiac silhouette.

Cardiac Tamponade Diagnosis and Pericardiocentesis Procedure

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA 
The heart lies within the pericardium, a ...

Lateral Canthotomy and Inferior Cantholysis

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Lateral canthotomy is a potentially eyesight-saving procedure when performed emergently for an orbital compartment syndrome. An orbital compartment syndrome results from a buildup of pressure behind the eye; as pressure mounts, both the optic nerve and its vascular supply are compressed, rapidly leading to nerve damage and blindness if the pressure is not quickly relieved.
The medial and lateral canthal tendons hold the eyelids firmly in place forming an anatomical compartment with limited space for the globe. In an orbital compartment syndrome, pressure rapidly increases as the globe is forced against the eyelids. Lateral canthotomy is the procedure by which the lateral canthal tendon is severed, thereby releasing the globe from its fixed position. Often, severing of the lateral canthal tendon alone is not enough to release the globe and the inferior portion (inferior crus) of the lateral canthal tendon also needs to be severed (inferior cantholysis). This increases precious space behind the eye by allowing the globe to become more proptotic, resulting in decompression. Most frequently, orbital compartment syndrome is the result of acute facial trauma, with the subsequent development of a retrobulbar hematoma.
Examination of the patient will reveal a proptotic globe as it strains from the pressure against the tendons anchoring it in place. Patients experience decreased visual acuity and severe eye pain. Patients may develop a relative afferent pupillary defect (RAFD), otherwise known as a Marcus Gunn pupil, and will have increased intraocular pressure (IOP).

Extraocular Eye: Lateral Canthotomy and Inferior Cantholysis

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Lateral canthotomy is a potentially eyesight-saving ...

Percutaneous Cricothyrotomy

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
A surgical airway procedure is indicated when other forms of endotracheal intubation have failed and ventilation is worsening or not possible. This is the feared &#34;can&#39;t intubate, can&#39;t ventilate&#34; scenario, and in the emergency setting, cricothyrotomy is the surgical procedure of choice.
Cricothyrotomy is preferred over tracheotomy because of the lower risk of complications, the predictable anatomy of the cricothyroid membrane, and the comparative rapidity with which the procedure can be performed&#8212;even by less experienced practitioners. Cricothyrotomy traditionally has been done in an &#34;open&#34; form; however, percutaneous cricothyrotomy using standard Seldinger technique has been advanced as a more successful approach when identification of the relevant anatomic landmarks is more difficult. Seldinger technique involves the introduction of a device into the body through the use of an introducer needle and a guide wire. The needle is used to locate the target; a guide wire is then fed through the thin-walled needle into the target, acting as a &#34;placeholder&#34; for the device, which is fed over the guide wire and into the target.
In the case of percutaneous cricothyrotomy, the practitioner first identifies the cricothyroid membrane by physical landmarks and makes a small vertical skin incision. A thin-walled 18-gauge needle (attached to a syringe) pierces the membrane, and the airway is positively identified when air is aspirated in the syringe. A guide wire is then fed through the needle. Standard cricothyrotomy sets include an airway catheter (similar to a tracheostomy tube) with a stiff dilator within its lumen. The catheter/dilator combination is fed onto the guide wire and the catheter/dilator is placed within the airway. The dilator and guide wire are subsequently removed, and the catheter is attached to a bag-valve device for ventilation.

Percutaneous Cricothyrotomy with and without a Kit

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
A surgical airway procedure is indicated when other ...

Open Cricothyrotomy

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Open cricothyrotomy is an emergent surgical procedure. It is performed to establish an airway access by passage of a tube through an incision in the cricothyroid membrane. This is a procedure of choice in the feared "can't intubate, can't ventilate" scenario - when all other forms of endotracheal intubation have failed and the spontaneous ventilation is worsening or has become impossible.
The airway access is established via the tracheostomy tube. The tracheostomy tube consists of three parts: an outer cannula (or the tracheostomy tube itself), an inner cannula, and an obturator. During the procedure, the obturator is placed within the tracheostomy tube to guide the insertion, while the inner cannula is removed. The distal end of the obturator is rounded and protrudes through the end of the tracheostomy tube, allowing the practitioner to easily guide the tube into place without it being caught on the surrounding structures. In addition, the obturator prevents the tube clogging with tissue or the fluids during an insertion. Once the tube is placed, the obturator is removed and the inner cannula is placed within the tracheostomy tube. Alternatively, the procedure can be performed using the modified the endotracheal tube, which will be demonstrated in this video.
Cricothyrotomy is associated with significant complications and is performed only when less invasive measures have failed. However, it is preferred over tracheotomy (a procedure in which an opening is created between two tracheal rings), because of the lower risk of associated complications, relative rapidity with which it can be performed, and the predictable anatomy of the region. 
Young age is considered a contraindication to open cricothyrotomy, as this procedure is associated with an increased risk of developing subglottic stenosis in children. However, there is disagreement among experts as to which age this procedure becomes acceptable. Opinions vary from 5 years to 12 years of age, and many consider this a relative contraindication. In young children, tracheotomy is preferred, and patients may be temporized with transtracheal jet ventilation through a needle cricothyrotomy for long enough to perform this more involved procedure. Other contraindications include fractured larynx or severe injury to the cricoid cartilage.

Open Cricothyrotomy: Airway Access via Tracheostomy Tube

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Open cricothyrotomy is an emergent surgical procedure. ...

Needle Thoracostomy

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
A tension pneumothorax is a life-threatening situation in which excess air is introduced into the pleural space surrounding the lung, either through trauma to the chest cavity or as a spontaneous leak of air from the lung itself. Air trapped within the pleural space causes separation of the lung from the chest wall, disrupting normal breathing mechanisms. Pneumothorax may be small without conversion to tension, but when there is a significant and expanding amount of air trapped in the pleural cavity, the increasing pressure from this abnormal air causes the lung to shrink and collapse, leading to respiratory distress. This pressure also pushes the mediastinum (including the heart and great vessels) away from its central position, causing inability of blood to return to the heart and diminishing the cardiac output. Tension pneumothoraces cause chest pain, extreme shortness of breath, respiratory failure, hypoxia, tachycardia, and hypotension. They need to be relieved emergently when a patient is in extremis.
Tension pneumothoraces are definitively managed by procedures that allow removal of trapped air, such as insertion of a chest tube. However, materials for chest tube placement are typically not available outside of the hospital setting. Temporizing measures are needed in deteriorating patients prior to hospital arrival or while chest tube materials are being gathered. In these situations, urgent needle thoracostomy (also called &#34;needle decompression&#34;) is performed. Simply, it is the insertion of a large-bore needle or cannula through the chest wall and into the pleural space to allow air within the pleural cavity to escape. If a catheter or cannula is not immediately available, the procedure may be performed with a long, large-bore needle attached to a syringe. Air can be aspirated out of the pleural space with the syringe. A metal needle cannot stay in the pleural cavity, as the sharp tip may cause further damage; thus, it would need to be removed from the chest wall once air is aspirated.

Pneumothorax and Needle Thoracostomy Procedure

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
A tension pneumothorax is a life-threatening ...

Tube Thoracostomy

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA 
Tube thoracostomy (chest tube placement) is a procedure during which a hollow tube is inserted into the thoracic cavity for drainage of fluid or air. Emergency chest tube insertion is performed for definitive treatment of tension pneumothorax, traumatic hemothorax, large-volume pleural effusions, and empyemas.
Irrespective of the cause of air and fluid accumulation in the pleural space, the drainage relieves lung compression and enables lung re-expansion. In pneumothorax, air accumulation in the pleural cavity separates pleural layers, which prevents lung expansion during the respiration. Abnormal fluid accumulation, such as in case of hemothorax or empyema, causes separation of the visceral pleura that adheres to lung tissue from the parietal pleura that forms the lining of the chest cavity. The uncoupling of the pleural layers leads to disconnection of chest wall movement from the lung movement, causing respiratory distress. In addition, excessive pressure from overwhelming amounts of air or fluid in the pleura may push the mediastinum away from the central chest, causing inability of blood to return to the heart.
In the trauma setting, a chest tube may not only treat a hemothorax but also allow monitoring of the bleeding rate. Massive hemothorax or continued brisk bleeding necessitates progression to a surgical thoracotomy, which is the opening of the chest wall to seal bleeding sites.
Chest tubes consist of clear plastic with a radiopaque strip running along their length, and fenestrations along the tip of the tube. Tube sizes vary from 12 to 42 French (Fr), with the smaller sizes used for pediatric cases. A size 36 Fr or larger is standard size to be used for hemothorax and empyema drainage.

Tube Thoracostomy: Chest Tube Placement Procedure

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA 
Tube thoracostomy (chest tube placement) is a ...

Intra-articular Shoulder Injection for Reduction Following Shoulder Dislocation

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
The anterior shoulder dislocation is one of the most common joint dislocations seen in emergency settings. In anterior shoulder dislocation, the humeral head is displaced out of the glenohumeral joint in front of the scapular glenoid, resulting in a loss of the articulation between the arm and the rest of the shoulder. This can be caused by a fall onto an abducted, extended, and externally rotated arm, such as in a bicycle or running accident. Sometimes anterior shoulder dislocation can be due to a minor trauma or even result from rolling over in bed with an externally rotated and stretched overhead arm.
Anterior shoulder dislocation is a painful injury. Patients cannot actively abduct, adduct or internally rotate the shoulder. Reduction of the shoulder is the best form of analgesia and, of course, is necessary to restore arm function. While it is current practice for patients to undergo procedural sedation during the shoulder reduction procedure, the sedatives have serious side effects (cardiac and respiratory depression), and require long stays in the emergency department (ED), dedicated nursing staff, multiple radiographs, and consulting services.
Intra-articular injection with a local anesthetic, such as lidocaine, offers significant pain relief in patients with shoulder dislocations and during the reduction procedure. It is a technically simple procedure that doesn&#39;t require long ED stays or significant hospital resources. The success of the intra-articular anesthesia can be enhanced further by performing the procedure under the guidance of bedside ultrasound, which allows real-time visualization of the needle tip reaching the appropriate area.

Intra-articular Shoulder Injection Following Shoulder Dislocation

Source: Rachel Liu, BAO, MBBCh, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
The anterior shoulder dislocation is one of the ...

Arterial Line Placement

Source: Sharon Bord, MD, Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
When monitoring patients, it is important to obtain values that are accurate and reliable. Blood pressure monitoring is one of the essential vital signs, and for a majority of patients, measuring it utilizing non-invasive techniques provides accurate values. However, there are situations in which the blood pressure requires more exact, specific, and reliable measurements. This can be achieved by intra-arterial blood pressure monitoring and requires arterial line placement. Arterial line placement refers to the insertion of a catheter, which is able to transduce blood pressure, into one of the major arteries (e.g., radial or femoral artery). Patients who potentially need arterial line placement include those with extreme low (such as in sepsis or cardiogenic shock) or high (as in cerebrovascular accident or hypertensive emergency) blood pressure measurements. Many of these patients are placed on vasoactive medications to either increase or decrease blood pressure. When the goal is to decrease a patient&#39;s blood pressure, it must be done gradually, which further necessitates close blood pressure monitoring. Arterial line placement is also ideal for patients who require frequent arterial blood gas monitoring.

Arterial Line Placement: Over-the-Wire and Over-the-Needle

Source: Sharon Bord, MD, Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
When monitoring patients, it is ...

Intraosseous Needle Placement

Source:&#160;<a href="https://www.jove.com/author/Julianna_Jung">Julianna Jung</a>, MD, FACEP, Associate Professor of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
For unstable patients requiring urgent administration of medications, fluids, or blood products, establishing vascular access quickly is essential. However, there are many factors that can complicate placement of a peripheral intravenous cannula (PIV), and it is extremely common for PIV attempts to fail. PIV placement may be technically challenging in small children, injection drug users, obese people, people with chronic illnesses necessitating frequent vascular access, and in those with burns and other skin conditions. Furthermore, for patients in shock, blood is shunted away from the periphery in order to compensate for impaired perfusion of vital organs, making peripheral vessels difficult to find and cannulate. In these situations, intraosseous (IO) needle placement is an extremely effective alternative to PIV placement, allowing rapid and technically straightforward access to the highly vascularized intramedullary space inside the long bones. From here, medications and fluids are readily absorbed into the bloodstream, permitting stabilization of critically ill patients.

Intraosseous Needle Placement Using a Intraosseous Drill

Source:&#160;Julianna Jung, MD, FACEP, Associate Professor of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
For ...

Peripheral Venous Cannulation

Source: Sharon Bord, MD, Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
Placement of an intravenous (IV) catheter is one of the key procedures in medicine. The IV catheter allows patients to receive critical medications, including pain medicine, insulin, antibiotics, blood products, and fluids for rehydration. Additionally, placing an IV catheter allows for blood samples to be obtained, which can be sent to the laboratory for testing and evaluation. A majority of peripheral IV lines are placed in the superficially located veins of the upper extremities. IV catheters can be placed in any superficial vein from the upper arm to the hand (though the veins in the antecubital fossa are larger than those in the hand). IV catheters can be placed in the lower extremities as well; however, this procedure should be performed with caution in patients with a history of diabetes or poor peripheral circulation.

Peripheral Venous Cannulation and Catheter Insertion

Source: Sharon Bord, MD, Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Maryland, USA
Placement of an intravenous (IV) ...

Central Venous Catheter Insertion: Internal Jugular

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Central venous access is necessary in a multitude of clinical situations, including vascular access, vasopressor and caustic medication delivery, central venous pressure monitoring, intravascular device delivery (pacing wires, Swann-Ganz catheters), volume resuscitation, total parental nutrition, hemodialysis, and frequent phlebotomy.
Safe reliable placement of a central venous catheter (CVC) in the internal jugular (IJ) vein using ultrasound guidance has become the standard of care. It is therefore imperative to understand the anatomy, the relationship between the IJ and the carotid artery, and their appearance on ultrasound. It is also necessary to have the psychomotor skills of vessel cannulation under ultrasound guidance.
Seldinger technique is an introduction of a device into the body over a guide wire, which is inserted through a thin-walled needle. In the case of CVC insertion, the device is an intravascular catheter and the target vessel is a central vein. First, the target vessel is cannulated with an 18 gauge thin-walled needle. A guide wire is then passed thought the needle until it is appropriately positioned within the vessel. The needle is removed, and a dilator is passed over the wire to dilate the skin and soft tissue to the level of the vessel. The dilator is then removed, and the catheter is passed over the wire until it is appropriately positioned within the vessel. Lastly, the guide wire is removed.
Successful placement of a CVC using ultrasound consists of a working understanding of the target anatomy, facility with procedural ultrasound, and fluidity in Seldinger technique. An IJ CVC can be placed in either the right or the left IJ vein. In general, however, the right IJ vein is preferred because of its straight route to the superior vena cava (SVC), which makes malposition of the catheter less likely.
There are several types of CVC kits commonly available marketed by different manufacturers. CVCs may a single lumen, a double lumen, or a triple lumen. For purposes of this discussion, we will place a triple-lumen CVC, as this is commonly needed when multiple different medications need to be delivered simultaneously. The procedure for placing any type of CVC is the same.

Catheter Insertion in Internal Jugular: the Seldinger Technique

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Central venous access is necessary in a multitude of ...

Central Venous Catheter Insertion: Femoral Vein

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Central venous access is necessary in a multitude of clinical situations, including vascular access, vasopressor and caustic medication delivery, central venous pressure monitoring, volume resuscitation, total parental nutrition, hemodialysis, and frequent phlebotomy. There are three veins in the body that are accessed for central venous cannulation: the internal jugular, the subclavian, and the femoral. Each of these vessels has distinct advantages and disadvantages with unique anatomical considerations. 
Femoral vein cannulation can be easily performed both under ultrasound guidance and using the surface landmarks; therefore, femoral access is often used when emergent placement of a central venous catheter (CVC) is needed (such as in the case of medical codes and trauma resuscitations). In addition, cannulation of the femoral artery allows one to simultaneously perform other procedures needed for stabilization, such as cardiopulmonary resuscitation (CPR) and intubation.
Successful placement of a femoral CVC requires working understanding of the target anatomy, access to with procedural ultrasound, and fluidity in the Seldinger technique.
Seldinger technique is the introduction of a device into the body over a guide wire, which is placed through a thin-walled needle. In the case of CVC insertion, the device is an intravascular catheter and the target vessel is a central vein. First, the target vessel is cannulated with an 18-gauge thin-walled needle. A guide wire is then passed through the needle until it is appropriately positioned within the vessel. The needle is removed and a dilator is passed over the wire to dilate the skin and soft tissue to the level of the vessel. The dilator is then removed and the catheter is passed over the wire until it is appropriately positioned within the vessel. Lastly, the guide wire is removed.
The main disadvantage of a femoral CVC is the high incidence of infection due to proximity to the groin (and often as a result of the quasi-sterile nature under which emergent CVCs are placed). Femoral lines should be replaced early on in the patient's hospital course if sustained central access is needed. A line placed with minimal sterility should be replaced as soon as the patient is stable.

Catheter Insertion in Femoral Vein: FV CVC Placement

Central Venous Catheter Insertion: Subclavian Vein

Source: James W Bonz, MD, Emergency Medicine, Yale School of Medicine, New Haven, Connecticut, USA
Central venous access is necessary in a multitude of clinical situations for hemodynamic monitoring, medication delivery, and blood sampling. There are three veins in the body that are accessed for central venous cannulation: the internal jugular, the subclavian, and the femoral vein.
Central venous access via the subclavian vein has several advantages over other possible locations. The subclavian central venous catheter (CVC) placement is associated with lower infection and thrombosis rate than internal jugular and femoral CVC. Subclavian line can be placed quickly using anatomic landmarks and are often performed in trauma settings when cervical collars obliterate the access to the internal jugular (IJ) vein. The most significant disadvantage of the subclavian access is the risk of pneumothorax due to the anatomic proximity to the dome of the lung, which lies just superficial to the subclavian vein. In addition, in the event of an inadvertent arterial puncture, the access to the subclavian artery is impeded by the clavicle, which makes it difficult to effectively compress the vessel.
Successful placement of the subclavian CVC requires good working understanding of the target vessel anatomy as well as fluidity in performing the Seldinger procedure (an introduction of the catheter into a vessel over the guide wire, which is inserted through a thin-walled needle). First, the subclavian vein is cannulated with an 18-gauge thin-walled needle. A guide wire is then passed thought the needle until it is appropriately positioned within the vessel. Next, the needle is removed, a dilator is passed over the wire to dilate the skin and soft tissue, and the catheter is passed over the wire until it is appropriately positioned within the vessel. Lastly, the guide wire is removed, and the catheter is sutured in place.
There are several types of CVC kits commonly available marketed by different manufacturers. CVCs may have a single lumen, a double lumen, or a triple lumen. For purposes of this discussion, we will place a triple-lumen CVC, as this is commonly needed when multiple different medications need to be delivered simultaneously. The procedure for placing any type of CVC is the same.

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