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 ...

General Approach to the Physical Exam

Source: <a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The examination of the body is fundamental to the practice of medicine. Since the Roman Empire, physicians have described the connection between alterations in function of specific parts of the body and specific disease states and have sought to further scientific understanding to improve bedside diagnosis. However, in this modern age of increasing technology within medical diagnostics, it is important to consider the role that physical examination plays today. It is misguided to believe that physical examination holds all the answers, and much has been written about the questionable utility of certain maneuvers previously held in high regard. It is equally misguided to suggest that physical examination plays little role in the modern patient encounter.
Physical examination remains a valuable diagnostic tool; there are many diagnoses that can only be made by physical examination. A diagnosis made by labs or imaging is rarely done in the absence of findings detectable at the bedside. As the provider conducts a history and physical, they are actively generating and testing hypotheses to explain the patient&#39;s condition. The information one gathers may not replace the need for testing, but having firm hypotheses in place allows the provider to order tests more judiciously and ask better questions of those tests. This, in turn, has the potential to reduce risk to the patient and save cost for the health care system. Finally, physical examination plays a critical role in the therapeutic relationship. By engaging in the time-honored ritual of physical examination, the clinician has the opportunity to develop rapport (by demonstrating attention and sensitivity) and promote healing through the deliberate and responsible use of touch.

Preliminary Diagnosis/Prognosis through Physical Examination

Source: Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The examination of the body is fundamental to ...

Observation and Inspection

Source: <a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Observation and inspection is fundamental to physical examination and begins at the first point of contact with a patient. While observation and inspection are often used interchangeably, observation is a general term that refers to the careful use of one&#39;s senses to gain information. Inspection is an act limited to what one can observe visually, and when referring to physical examination, typically refers to findings on the surface of the body, rather than to behaviors. Skilled clinicians utilize all of their senses to assist with gaining an understanding of their patients, relying on vision, touch (percussion and palpation), and hearing (percussion and auscultation) primarily. Smell can also provide important diagnostic information during the patient encounter (e.g., personal hygiene, substance use, or metabolic diseases). Fortunately the sense of taste is largely a historical relic in medicine, though it is interesting to note that diabetes mellitus was diagnosed for many centuries by the sweet taste of the urine. Through experience, clinicians develop an important sixth sense - the gut instinct - that can only be gained through deliberate practice of clinical skills on thousands of patients over many years. The clinician&#39;s gut instinct, which is based largely on bedside observations, has been shown to be a strong predictor of serious illness. This video and the others in the clinical skills video collection are steps on the way to learning this level of mastery.

Inspection of the Skin, Eyes, Ears, Scalp and Medical History

Source: Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Observation and inspection is fundamental to ...

Palpation

Source: <a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The physical examination requires the use of all of the provider&#39;s senses to gain information about the patient. The sense of touch is utilized to obtain diagnostic information through palpation.
The specific parts of the examiner&#39;s hand used for palpation differ based on the body part being examined. Because of their dense sensory innervation, the finger pads are useful for fine discrimination (e.g., defining the borders of masses, lymph nodes) (Figure 1). The dorsal surface of the hand provides a rough sense of relative temperature (Figure 2). The palmar surfaces of the fingers and hands are most useful for surveying large areas of the body (e.g., abdomen) (Figure 3). Vibration is best appreciated with the ulnar surface of the hands and 5th fingers (e.g., tactile fremitus) (Figure 4).
While palpation is fundamental to the diagnostic aspect of the physical exam, it is also important to acknowledge the role that touch plays in communicating caring and comfort during the patient encounter. Patients generally perceive touch from a healthcare provider in a positive light, and their perceptions of a healthcare provider can be shaped by the skilled use of touch during clinical encounters.1 Physical contact has been associated with alterations in hormonal and neurotransmitter levels, specifically decreases in cortisol and increases in serotonin.2
Therefore, through the careful use of palpation, and touch in general, during the physical examination, the clinician has the opportunity to gain important diagnostic information, while developing rapport and promoting healing.
<img alt="Figure 1" src="/files/ftp_upload/10143/10143fig1.jpg" /> 
Figure 1. Lymph node examination with finger pads. 
<img alt="Figure 2" src="/files/ftp_upload/10143/10143fig2.jpg" /> 
Figure 2. Using the dorsum of hand to assess for warmth.
<img alt="Figure 3" src="/files/ftp_upload/10143/10143fig3.jpg" /> 
Figure 3. Abdominal palpation with palmar surface of fingers and hands. 
<img alt="Figure 4" src="/files/ftp_upload/10143/10143fig4.jpg" /> 
Figure 4. Using the ulnar surface of the hand to assess for tactile fremitus.

Palpation for Lymph Nodes, Abdomen and Tactile Fremitus

Source: Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The physical examination requires the use of ...

Percussion

Source: <a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Simply stated, percussion refers to the striking of one object against another to produce sound. In the early 1700s, an Austrian inn-keeper&#39;s son, named Leopold Auenbrugger, discovered that he could take inventory by tapping his father&#39;s beer barrels with his fingers. Years later, while practicing medicine in Vienna, he applied this technique to his patients and published the first description of the diagnostic utility of percussion in 1761. His findings faded into obscurity until the prominent French physician Jean-Nicolas Corvisart rediscovered his writings in 1808, during an era in which great attention was focused on diagnostic accuracy at the bedside.1
There are three types of percussion. Auenbrugger and Corvisart relied on direct percussion, in which the plexor (i.e. tapping) finger strikes directly against the patient&#39;s body. An indirect method is used more commonly today. In indirect percussion, the plexor finger strikes a pleximeter, which is typically the middle finger of the non-dominant hand placed against the patient&#39;s body. As the examiner&#39;s finger strikes the pleximeter (or directly against the surface of the patient&#39;s body), sound waves are generated. If using indirect percussion, important information is gained from the vibration in the pleximeter finger, as well.2 The third type of percussion, auscultatory percussion, relies on the clinician using a stethoscope to discern differences in sounds created by the plexor finger.
The density of the structure underlying the site of percussion determines the tone of the percussion note; the denser the structure, the quieter the note. Notes differ in relative intensity, pitch, and duration, and help the examiner determine what lies below the skin surface. Knowledge of what particular locations on the body should sound like, in conjunction with the particulars of a specific clinical situation, can help a clinician determine if percussion notes in a particular patient are normal or not.

Clinical Percussion Examination: Direct, Indirect and Auscultatory

Source: Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Simply stated, percussion refers to the ...

Auscultation

Source: <a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Through auscultation, the clinician is able &#34;to eavesdrop on the workings of the body&#34; to gain important diagnostic information.1 Historically, the term &#34;auscultation&#34; was synonymous with &#34;immediate auscultation,&#34; in which the examiner&#39;s ear was placed directly against the patient&#39;s skin. Although this was standard practice for centuries, the method proved inadequate in nineteenth-century France, due to social norms and suboptimal diagnostic yield. This led Ren&#233; La&#235;nnec to invent the first stethoscope in 1816 (Figure 1), a tool that has since become inseparable from auscultation in modern clinical practice, and patients hold it as a symbol of honor and trustworthiness among those who carry them.2
<img alt="Figure 1" src="/files/ftp_upload/10153/10153fig1.jpg" /> 
Figure 1. A representative illustration of the first stethoscope invented by Ren&#233; La&#235;nnec.
The stethoscope has undergone many technologic advances since La&#235;nnec&#39;s initial hollow wooden tube. Practically speaking, the provider must understand the difference between the two sides of the modern stethoscope&#39;s chest piece: the diaphragm and the bell (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/10153/10153fig2.jpg" /> 
Figure 2. Parts of a modern stethoscope.
When applied firmly against the patient&#39;s skin, the diaphragm transmits high frequency sounds. Sounds from within the patient vibrate the membrane of the diaphragm. These vibrations result in the propagation of sound through the column of air inside the stethoscope and into the examiner&#39;s ears. Conversely, when applied lightly, the bell transmits low frequency sounds. The bell acts as a cup that directly transmits sounds from within the patient through the tubing of the stethoscope. Pressing more firmly with the bell can stretch the underlying skin, essentially turning it into a diaphragm. Auscultation is used in a wide variety of clinical settings. It most commonly plays a role in the examination of the chest, heart, abdomen, and vasculature.

Auscultation and Working Mechanism of a Stethoscope

Source: Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Through auscultation, the clinician is able ...

Proper Adjustment of Patient Attire during the Physical Exam

Source: <a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, and Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
In order to optimize the predictive value of the physical examination, the provider must perform maneuvers correctly. The proper use of drapes is an important component of correctly performing physical examination maneuvers. Skin lesions are missed when &#34;inspection&#34; occurs through clothing, crackles are erroneously reported when the lungs are examined through a t-shirt, and subtle findings on the heart exam go undetected when auscultation is performed over clothing. Accordingly, the best practice standards call for examining with one&#39;s hands or equipment in direct contact with the patient&#39;s skin (i.e., do not examine through a gown, drape, or clothing). In addition to its clinical value, the correct draping technique is important for improving the patient&#39;s comfort level during the encounter.
Like all other aspects of the physical exam, it takes deliberate thought and practice to find the right balance between draping, which is done to preserve patient modesty, and exposure, which is necessary to optimize access to the parts that need examination. Individual provider styles in the use of gowns and drapes vary considerably based on the site of practice, resource availability, and discipline within medicine. This video provides a general overview of some of the most common techniques used, combining techniques that utilize common draping approaches.

Patient Attire During Physical Exam; Use of Gowns and Drapes

Source: Jaideep S. Talwalkar, MD, and Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
In order to optimize the ...

Blood Pressure Measurement

Source: Meghan Fashjian, ACNP-BC, Beth Israel Deaconess Medical Center, Boston MA
The term blood pressure (BP) describes lateral pressures produced by blood upon the vessel walls. BP is a vital sign obtained routinely in hospital and outpatient settings, and is one of the most common medical assessments performed around the world. It can be determined directly with the intra-arterial catheter or by indirect method, which is a non-invasive, safe, easily reproducible, and thus most used technique. One of the most important applications of BP measurements is the screening, diagnosis, and monitoring of hypertension, a condition that affects almost one third of the U.S. adult population and is one of the leading causes of the cardiovascular disease.
BP can be measured automatically by oscillometry or manually by auscultation utilizing a sphygmomanometer, a device with an inflatable cuff to collapse the artery and a manometer to measure the pressure. Determination of the pulse-obliterating pressure by palpation is done prior to auscultation to give a rough estimate of the target systolic pressure. Next, the examiner places a stethoscope over the brachial artery of the patient, inflates the cuff above the expected systolic pressure, and then auscultates while deflating the cuff and observing the manometer readings. When the pressure in the cuff falls below the pressure in the brachial artery, the turbulent blood flow in a partially squeezed artery produces the Korotkoff audible sounds. The first audible Korotkoff sound signifies the maximum arterial pressure during systole. When the pressure in the cuff is reduced further and falls below the minimal arterial pressure (during diastole), the Korotkoff sounds become no longer audible. The reading at this point signifies the diastolic blood pressure. The blood pressure is measured in mmHg and recorded as a fraction (systolic BP/ diastolic BP).
In most instances, the vital signs are initially measured by a health care assistant or registered nurse (RN). The physician may choose to repeat the vital signs and blood pressure measurement after completion of the patient interview. Repeated measurement of blood pressure is especially important given the potential measurement errors and blood pressure variations.

Blood Pressure Measurement Using a Sphygmomanometer

Source: Meghan Fashjian, ACNP-BC, Beth Israel Deaconess Medical Center, Boston MA
The term blood pressure (BP) describes lateral pressures produced by ...

Measuring Vital Signs

Source: Meghan Fashjian, ACNP-BC, Beth Israel Deaconess Medical Center, Boston MA
The vital signs are objective measurements of a patient&#39;s clinical status. There are five commonly accepted vital signs: blood pressure, heart rate, temperature, respiratory rate, and oxygen saturation. In many practices, pain is considered the sixth vital sign and should regularly be documented in the same location as the other vital signs. However, the pain scale is a subjective measurement and, therefore, has a different value according to each individual patient.
The vital signs assessment includes estimation of heart rate, blood pressure (demonstrated in a separate video), respiratory rate, temperature, oxygen saturation, and the presence and severity of pain. The accepted ranges for vital signs are: heart rate (HR), 50-80 beats per minute (bpm); respiratory rate (RR), 14-20 bpm; oxygen saturation (SaO2), &#62; 92%; and average oral temperature, ~98.6 &#176;F (37 &#176;C) (average rectal and tympanic temperatures are ~1&#176; higher, and axillary temperature is ~1&#176; lower compared to the average oral temperature).
Vital signs serve as the first clue that something may be amiss with a patient, especially if the patient is unable to communicate. Although there are quoted normal ranges, each patient should be considered as an individual and not treated without taking into account the entire clinical picture.

Vital Signs: BP, Heart Rate, Temperature, Respiratory Rate and Oxygen Saturation

Source: Meghan Fashjian, ACNP-BC, Beth Israel Deaconess Medical Center, Boston MA
The vital signs are objective measurements of a patient&#39;s clinical ...

Respiratory Exam I: Inspection and Palpation

Source: <a href="https://www.jove.com/author/Suneel_Dhand">Suneel Dhand</a>, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Disorders of the respiratory system with a chief complaint of shortness of breath are among the most common reasons for both outpatient and inpatient evaluation. The most obvious visible clue to a respiratory problem will be whether the patient is displaying any signs of respiratory distress, such as fast respiratory rate and/or cyanosis. In a clinical situation, this will always require emergent attention and oxygen therapy.
Unlike pathology in other body systems, many pulmonary disorders, including chronic obstructive pulmonary disease (COPD), asthma, and pneumonia, can be diagnosed by careful clinical examination alone. This starts with a comprehensive inspection and palpation. Keep in mind that in non-emergency situations the patient&#39;s complete history will have been taken already, gaining important insight into exposure histories (e.g., smoking), which could give rise to specific lung diseases. This history can then confirm physical findings as the examination is performed.

Respiratory Exam I: Palpation, Chest and Peripheral Examination

Source: Suneel Dhand, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Disorders of the respiratory system with a chief ...

Respiratory Exam II: Percussion and Auscultation

Source: <a href="https://www.jove.com/author/Suneel_Dhand">Suneel Dhand</a>, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Learning the proper technique for percussion and auscultation of the respiratory system is vital and comes with practice on real patients. Percussion is a useful skill that is often skipped during everyday clinical practice, but if performed correctly, it can help the physician to identify underlying lung pathology. Auscultation can provide an almost immediate diagnosis for a number of acute pulmonary conditions, including chronic obstructive pulmonary disease (COPD), asthma, pneumonia, and pneumothorax.
The areas for auscultating the lungs correspond to the lung zones. Each lung lobe can be pictured underneath the chest wall during percussion and auscultation (Figure 1). The right lung has three lobes: the superior, middle, and inferior lobes. The left lung has two lobes: the superior and inferior lobes. The superior lobe of the left lung also has a separate projection known as the lingual.
<img alt="Figure 3" src="/files/ftp_upload/10041/10041fig1.jpg" /> 
Figure 1. Anatomy of lungs with respect to the chest wall. An approximate projection of lungs and their fissures and lobes to the chest wall anteriorly. RUL - right upper lobe; RML - right middle lobe; RLL - right lower lobe; LUL - left upper lobe; LLL - left lower lobe.

Respiratory Exam II: Percussion, Auscultation, Lung Lobes and Breath Sounds

Source: Suneel Dhand, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Learning the proper technique for percussion and ...

Cardiac Exam I: Inspection and Palpation

Source: <a href="https://www.jove.com/author/Suneel_Dhand">Suneel Dhand</a>, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
The cardiac assessment is one of the core examinations performed by almost every physician whenever encountering a patient. Disorders of the cardiac system are among the most common reasons for hospital admission, with conditions ranging from myocardial infarction to congestive heart failure. Learning a complete and thorough cardiac examination is therefore crucial for any practicing physician.
If there is pathology in the heart or circulatory system, the consequences can also be manifested in other bodily areas, including the lungs, abdomen, and legs. Many physicians instinctively reach straight for their stethoscopes when performing cardiac exams. However, a large amount of information is gained before auscultation by going through the correct sequence of examination, starting with inspection and palpation.

Cardiac Exam I: Palpation, Chest and Peripheral Examination

Source: Suneel Dhand, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
The cardiac assessment is one of the core ...

Cardiac Exam II: Auscultation

Source: <a href="https://www.jove.com/author/Suneel_Dhand">Suneel Dhand</a>, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Proficiency in the use of a stethoscope to listen to heart sounds and the ability to differentiate between normal and abnormal heart sounds are essential skills for any physician. Correct placement of the stethoscope on the chest corresponds to the sound of cardiac valves closing. The heart has two main sounds: S1 and S2. The first heart sound (S1) occurs as the mitral and tricuspid valves (atrioventricular valves) close after blood enters the ventricles. This represents the start of systole. The second heart sound (S2) occurs when the aortic and pulmonary valves (semilunar valves) close after blood has left the ventricles to enter the systemic and pulmonary circulation systems at the end of systole. Traditionally, the sounds are known as a &#34;lub-dub.&#34;
Auscultation of the heart is performed using both diaphragm and bell parts of the stethoscope chest piece. The diaphragm is most commonly used and is best for high-frequency sounds (such as S1 and S2) and murmurs of mitral regurgitation and aortic stenosis. The diaphragm should be pressed firmly against the chest wall. The bell best transmits low-frequency sounds (such as S3 and S4) and the murmur of mitral stenosis. The bell should be applied with a light pressure.

Cardiac Exam II: Auscultation and Lub-dub Heart Sounds

Source: Suneel Dhand, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Proficiency in the use of a stethoscope to listen ...

Cardiac Exam III: Abnormal Heart Sounds

Source: <a href="https://www.jove.com/author/Suneel_Dhand">Suneel Dhand</a>, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Having a fundamental understanding of normal heart sounds is the first step toward distinguishing the normal from the abnormal. Murmurs are sounds that represent turbulent and abnormal blood flow across a heart valve. They are caused either by stenosis (valve area too narrow) or regurgitation (backflow of blood across the valve) and are commonly heard as a &#34;swishing&#34; sound during auscultation. Murmurs are graded from 1 to 6 in intensity (1 being the softest and 6 the loudest) (Figure 1). The most common cardiac murmurs heard are left-sided murmurs of the aortic and mitral valves. Right-sided murmurs of the pulmonary and tricuspid valves are less common. Murmurs are typically heard loudest at the anatomical area that corresponds with the valvular pathology. Frequently, they also radiate to other areas.
<img alt="Figure 1" src="/files/ftp_upload/10135/10135fig1.jpg" /> 
Figure 1. The Levine scale used to grade murmur intensity. 
In addition to the two main heart sounds, S1 and S2, which are normally produced by the closing of heart valves, there are two other abnormal heart sounds, known as S3 and S4. These are also known as gallops, because of the &#34;galloping&#34; nature of more than two sounds in a row. S3 is a low-pitched sound heard in early diastole, caused by blood entering the ventricle. S3 is a sign of advanced heart failure, although it can be normal in some younger patients. S4 is heard in late diastole and represents ventricular filling due to atrial contraction in the presence of a stiff ventricle. S4 is also heard in heart failure and left ventricular hypertrophy.

Cardiac Exam III: Abnormal Heart Sounds and Levine Scale

Source: Suneel Dhand, MD, Attending Physician, Internal Medicine, Beth Israel Deaconess Medical Center
Having a fundamental understanding of normal heart ...

Peripheral Vascular Exam

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The prevalence of peripheral vascular disease (PVD) increases with age and is a significant cause of morbidity in older patients, and peripheral artery disease (PAD) is associated with cardiovascular and cerebrovascular complications. Diabetes, hyperlipidemia, hypertension, and tobacco use are important disease risk factors. When patients become symptomatic, they frequently complain of limb claudication, defined as a cramp-like muscle pain that worsens with activity and improves with rest. Patients with chronic venous insufficiency (CVI) often present with lower extremity swelling, pain, skin changes, and ulceration.
While the benefits of screening asymptomatic patients for PVD are unclear, physicians should know the proper exam technique when the diagnosis of PVD is being considered. This video reviews the vascular examination of the upper and lower extremities and abdomen. As always, the examiner should use a systematic method of examination, though in practice, the extent of the exam a physician performs depends on their suspicion of underlying PVD. In a patient who has or is suspected to have risk factors for vascular disease, the vascular exam should be thorough, beginning with inspection, followed by palpation, and then auscultation, and it should include special maneuvers, such as determining the ankle brachial index. Maneuvers that make use of a handheld Doppler are demonstrated in a companion video.
<img alt="Figure 1" src="/files/ftp_upload/10122/10122fig1.jpg" /> 
Figure 1. The major arm and leg arteries.

Peripheral Vascular Exam and Major Arm and Leg Arteries

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The prevalence of peripheral vascular disease (PVD) ...

Peripheral Vascular Exam Using a Continuous Wave Doppler

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Peripheral vascular disease (PVD) is a common condition affecting older adults and includes disease of the peripheral arteries and veins. While the history and physical exam offer clues to its diagnosis, Doppler ultrasound has become a routine part of the bedside vascular examination. The video titled &#34;The Peripheral Vascular Exam&#34; gave a detailed review of the physical examination of the peripheral arterial and venous systems. This video specifically reviews the bedside assessment of peripheral arterial disease (PAD) and chronic venous insufficiency using a handheld continuous wave Doppler.
The handheld Doppler (HHD) is a simple instrument that utilizes continuous transmission and reception of ultrasound (also referred to as continuous wave Doppler) to detect changes in blood velocity as it courses through a vessel. The Doppler probe contains a transmitting element that emits ultrasound and a receiving element that detects ultrasound waves (Figure 1). The emitted ultrasound is reflected off of moving blood and back to the probe at a frequency directly related to the velocity of blood flow. The reflected signal is detected and transduced to an audible sound with a frequency directly related to that of the received Doppler signal (thus, faster blood flow produces a higher frequency sound).
<img alt="Figure 1" src="/files/ftp_upload/10123/10123fig1.jpg" /> 
Figure 1. Generation of a Doppler signal. The handheld Doppler emits an ultrasound signal, which is then reflected back by moving blood, and finally received by the Doppler probe.
The HHD is easily used in the office or hospital setting to detect pulses, screen for PAD using the ankle brachial pressure index (ABPI), and localize venous insufficiency. This video reviews these procedures; however, it is not intended to be a comprehensive review of non-invasive vascular testing.

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