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JoVE Behavior documents observational and experimental techniques that seek to understand human and animal behavior. Work in this field includes analysis of genetic causes and physiological responses to internal or external stimuli as well as the implicated brain regions.

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JoVE Biochemistry publishes experiments that investigate the structure, function, and interactions of biological molecules. The video articles in this section demonstrate methods to study nucleic acids, proteins, carbohydrates and lipids as well as enzyme function, protein folding, molecular interactions and biochemical reactions.

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JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

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JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.

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JoVE Cancer Research illustrates experimental approaches in biomedical research and clinical practice aimed at understanding, detecting, treating and preventing cancer. The video articles in this section demonstrate methods to study the cellular and molecular mechanisms of cancer, animal models of cancer research, development of new therapies and diagnostics, tumor imaging, chemical and radiation treatments, surgical procedures and improvements in therapy. These video articles offer researchers and clinical practitioners an efficient way to learn the technologies and experimental methods in cancer research.

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JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

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JoVE Environment is dedicated to research methodologies that address environmental concerns and seek to better understand Earth's ecosystem.  Special consideration is given to experimental methodologies that assess society’s impact on the environment, suggest solutions for protecting Earth’s resources, and develop sustainable fuel sources. Moving towards an environmentally conscious planet is a collaborative effort with contributions from both life and physical scientists.

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JoVE Medicine serves as a connection between basic research and clinical applications. Articles may present medical procedures, case studies or clinical trial methodologies. Included studies are often modeled towards predictive, preventative, personalized, and practical approaches to treatment of disease. The goal of research methodologies in this section is to improve overall patient care and prognosis.

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JoVE Neuroscience is a multidisciplinary section devoted to investigations of the structure, function, physiology, and pathophysiology of the brain and nervous system. Included methodologies range from molecular and cellular level studies to full central and peripheral neural systems. Potential treatment platforms and surgical techniques for neurological diseases and disorders are also presented in this section.

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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 ...

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 ...

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 ...

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 ...

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 Labile Protons: Temporal Resolution

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

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

&sup1;H NMR of Conformationally Flexible Molecules: 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 ...

Chemical Ionization (CI) Mass Spectrometry

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

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

Mass Spectrometry: Alkyl Halide Fragmentation

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

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

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: 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 ...

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 ...

Mass Spectrometry: Alkyne Fragmentation

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

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

Mass Spectrometry: Aromatic Compound Fragmentation

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

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

Mass Spectrometry: Cycloalkene Fragmentation

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

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

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

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

The reaction proceeds via the cleavage of two ...

Mass Spectrometry: 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: 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 ...

From Theory to Design: The Role of Creativity in Designing Experiments

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

Research studies come into being when a researcher speculates about human thought, emotions, or behavior, and has a theory that offers a potential explanation. Often the researcher&#8217;s theory is firmly situated in everyday common experiences that may not naturally lend themselves to direct empirical study. 
 
For example, researchers speculated that perception of a person on Facebook is influenced by the appearances and comments of the person&#8217;s Facebook friends.1 It is difficult to test this theory using real-life Facebook profiles. Instead, researchers must use their creativity to design a study&#8212;in this case, using fake profiles that look highly realistic&#8212;to test their theory.&#160;

This video demonstrates how researchers test a central tenet of a popular social psychology theory. Specifically, this video shows a test of whether engaging in a self-expanding activity leads a person to feel a greater sense of self-efficacy.2

Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented, i.e., the margin of error accompanied by studying human behavior is sufficiently accounted for. In this video, we demonstrate this experiment using just 2 participants, one for each condition. However, as represented in the results, we used a total of 100 (50 for each condition) participants to reach the experiment&#8217;s conclusions.

从理论到设计： 创意设计实验中的作用

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

Research studies come into being when a ...

Ethics in Psychology Research

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

When a researcher finds an interesting topic to study such as aggression, the goal is often to study it in a way that is as true to life as possible. However, researchers must act in an ethical manner.&#160; To do this, they must balance their research goals with the best interests of the participants. Ethics often enter into the planning process when researchers identify all of the ways they can manipulate or measure a variable, but then make their final decision based on how they should manipulate or measure a variable.
After receiving a poor grade on a test or paper, a college student may appear to take it out on (i.e., act in an aggressive manner toward) their roommates by being mean or nasty, screaming, throwing things, or even becoming physically violent. Aggression is an important human behavior to study and understand due to the implications it has for interpersonal violence. However, for safety reasons, a study cannot expose participants to the risk that serious types of violence presents. As a result, researchers must identify similar but benign behaviors that can help us understand more aggressive behaviors without harming participants.
This video uses a two-group experiment to see if people really take out their anger on others even though the others are not responsible for the original problem. Specifically, it examines whether negative feedback leads to aggression while considering key ethical issues such as harm to participants, costs vs. benefits, informed consent, and debriefing.
Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to better ensure that the population under study is better represented, i.e., the margin of error accompanied by studying human behavior is sufficiently accounted for. In this video we demonstrate this experiment using just two participants, one being the evaluator. However, as represented in the results, we used a total of 245 participants to reach the experiment&#8217;s conclusions.

Ethical Principles in Designing Psychological Experiments

在心理学研究中的伦理

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

When a researcher finds an interesting topic to ...

Realism in Experimentation

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In an ideal world researchers would conduct their studies in real world settings where behaviors naturally happen. For example, if you want to see what influences individuals&#8217; voting behavior, it would be best to watch them vote. However, research in these settings is not always ethical or even practical. Further, a researcher may want more control over the setting to better pinpoint the exact variables that are influencing an outcome.&#160;
When researchers need to conduct studies in a lab, they try to optimize mundane realism, which means that they do everything they can to make the lab feel like a real-life experience. This video demonstrates a two-group design that examines how researchers use mundane realism in a lab to determine whether positive restaurant reviews are connected to diners&#8217; level of tipping.
Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented and the margin of error accompanied by studying human behavior is sufficiently accounted for.
In this video, we demonstrate this experiment using just two participants, one for each condition. However, as represented in the results, we used a total of 200 (100 for each condition) participants to reach the experiment&#8217;s conclusions.

在实验中的现实主义

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In an ideal world researchers would conduct ...

Perspectives on Experimental Psychology

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

As the Department Chair of Psychology at Monmouth University, I teach primarily upper level undergraduate students who are interested in Experimental Methods. These classes don&#8217;t have the same inherent appeal to students. This JoVE Psychology collection will help students vicariously watch the experiments being performed from start to finish, allowing them to gain exposure to over a dozen topics that they may not experience otherwise. Importantly, the videos provide a context for seeing the experiments embedded in their natural states&#8212;in the actual research process.
A few projects in this Science Education collection stand out. First, I consider the Ethics in Psychological Research video to be very useful in teaching, as it demonstrates how sensitive topics, such as interpersonal violence, can be studied in creative ways. Furthermore, students learn better with topics that they care about and can conceptualize in their personal lives. For example, Observational Research delves into the factors that go into homesickness, going beyond questionnaires and examining the relationship to what individuals actually leave around in their rooms. This design allows the researcher to curiously discover conclusions that students may not be fully aware of themselves.
Being an instructor and textbook author of research methods, I am keenly aware of the relative lack of quality resources that are available to teach Experimental Psychology well. These JoVE videos will help bring the material to life for instructors and particularly for students who do not necessarily get the opportunity to complete their own research projects.&#160;

实验心理学的观点

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

As the Department Chair of Psychology at ...

Pilot Testing

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In any experiment researchers have the challenge of creating experiences for participants that are consistent (i.e., reliable) and authentic (i.e., valid). Yet there are many ways to manipulate any one variable. For example, if you want participants to feel sad, you can have them think of their own sad memory, watch a sad video, or read a sad story.
Researchers must find the best way to operationalize a psychological construct in order to produce the most effective manipulation possible. Often, before running the main study, researchers will pilot test (i.e., try out) their manipulations to check their effectiveness.
This video demonstrates how to operationalize the same independent variable (acute stress) in three different ways. Specifically, this study seeks to identify the best sound (static, ticking clock, or crying baby) to play during a difficult task (solving complex math problems) to optimally manipulate stress.
Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented and the margin of error accompanied by studying human behavior is sufficiently accounted for. In this video, we demonstrate this experiment using just three participants, one for each condition. However, as represented in the results, we used a total of 120 (40 for each condition) participants to reach the experiment&#8217;s conclusions reflected in the Results section.

Conducting Pilot Testing in Psychological Research

试点测试

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In any experiment researchers have the challenge ...

Observational Research

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

If you want to know how someone thinks or feels, you can ask that person questions. &#160;Another approach is to observe how the person is acting or look for indicators of how they acted in the past. While observations may seem revealing, it isn&#8217;t always easy to know if they are truly accurate. For instance, you may see a person smiling and assume they are happy, when in reality they are annoyed and merely being polite.
The purpose of science is to move beyond an individual&#8217;s own views of the self because they are inherently skewed by that individual&#8217;s expectations, previous experience, personal biases, motivations, emotions, etc. While a person may have unique insight into one&#8217;s self, these insight may not accurately represent reality. Put more simply, what a person says, does not always match up well with what they actually do. For this reason, researchers should incorporate a variety of measures (e.g., asking participants to report how they feel, but also observing actual behavior) in order to more accurately capture how the person truly feels.
This video demonstrates a correlational design where researchers measure students&#8217; homesickness in two distinct ways: (1) a homesickness scale, and (2) by observing how the student has decorated his or her dorm room.&#160;
Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented, i.e., the margin of error accompanied by studying human behavior is sufficiently accounted for. In this video, we demonstrate this experiment using just one participant. However, as represented in the results, we used a total of 63 participants to reach the experiment&#8217;s conclusions.

Observational Research: Observations and Correlations

观察性研究

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

If you want to know how someone thinks or feels, ...

The Simple Experiment: Two-group Design

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A two-group design is the simplest way to establish a cause-effect relationship between two variables. This video demonstrates a simple experiment (two-group design).&#160; In providing an overview of how a researcher conducts a simple experiment (two-group design), this video shows viewers the process of turning ideas into testable ideas and forming hypothesis, the identification and effect of experiment variables, the formation of experimental conditions and controls, the process of conducting the study, the collection of results, and the consideration their implications. This research technique is demonstration in the context of answering the research question: &#8220;How does physiological arousal/excitement influence perceived attraction?&#8221;

Two-group Design: Cause-effect Relationship in Psychology Experiments

简单的实验： 两组设计

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A two-group design is the simplest way to ...

The Multi-group Experiment

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A multi-group design is an experimental design that has 3 or more conditions/groups of the same independent variable. This video demonstrates a multi-group experiment that examines how different interethnic ideologies (multiculturalism and color-blind) influence feelings about diversity and actions toward and out-group member. In providing an overview of how a researcher conducts a multi-group experiment, this video shows viewers how to distinguish levels in variables, common types of conditions/groups to use (including placebo and empty-control conditions/groups), the process of conducting the study, the collection of results, and the consideration of their implications.&#160; &#160;

Designing and Conducting Multi-group Experiment

多组实验

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A multi-group design is an experimental design ...

Within-subjects Repeated-measures Design

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A within-subjects, or repeated-measures, design is an experimental design where all the participants receive every level of the treatment, i.e., every independent variable. For example, in a candy taste test, the researcher would want every participant to taste and rate each type of candy.

This video demonstrates a within-subjects experiment (i.e., one where there is an independent variable with several variations or levels) that examines how different motivational messages (e.g., hard work, self-affirmation, outcomes, and positive affect) influence willingness to exert physical effort. As a within-subjects design, the participant will read each of the four types of motivational messages and then lift weights to measure physical effort. By providing an overview of how a researcher conducts a repeated-measures experiment, this video allows viewers to see how to address order effects through counterbalancing, which involves a systematic approach to making sure all possible orders of the conditions occur in the study.

Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented and the margin of error accompanied by studying human behavior is sufficiently addressed. In this video, we demonstrate this experiment using just one participant. However, as represented in the results, we used a total of 72 participants to reach the experiment&#8217;s conclusions.

Within-subjects/ Repeated-measures Design

内的主题重复测量设计

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A within-subjects, or repeated-measures, design ...

The Factorial Experiment

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A factorial design is a common type of experiment where there are two or more independent variables. This video demonstrates a 2 x 2 factorial design used to explore how self-awareness and self-esteem may influence the ability to decipher nonverbal signals. This video leads students through the basics of a factorial design including, the nature of a factorial design and what distinguishes it from other designs, the benefits of factorial design, the importance and nature of interactions, main effect and interaction hypotheses, and how to conduct a factorial experiment.

Designing and Conducting 2X2 Factorial Experiments

析因实验

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

A factorial design is a common type of ...

Self-report vs. Behavioral Measures of Recycling

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

One of the challenges in measuring an experimental variable is identifying the technique that will produce the more accurate measurement. The most common way to measure a dependent variable is self-report&#8212;asking the participant to describe his/her feelings, thoughts, or behaviors. Yet, people may not be honest. To truly know something about a participant, it may be necessary to see what they actually do in a situation.
This video uses a multi-group experiment to see if feeling close to others results in more favorable attitudes toward environmental consciousness measured both by self-report and behavioral observation.
Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to better ensure that the population under study is better represented, i.e., the margin of error accompanied by studying human behavior is sufficiently accounted for. In this video, we demonstrate this experiment using just one participant. However, as represented in the results, we used a total of 186 participants to reach the experiment&#8217;s conclusions.

Comparing Self-report Surveys and Behavioral Observations

自我报告与行为措施的回收

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

One of the challenges in measuring an ...

Reliability in Psychology Experiments

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In order to study something scientifically, a researcher needs to determine a way to quantify it. However, psychological constructs can be challenging to measure and quantify. This video examines reliability in the context of content analysis.&#160;
A recent study in the journal Pediatrics reported that 4-year-olds who watched a fast-paced cartoon had worse performance on cognitive tasks, such as following rules in a game, listening to direction from an adult, and delaying gratification, compared to other children who watched a slower paced cartoon.1 In addition to the pace of the cartoon, the content of the cartoon may also have deleterious effects on its young viewers.
This video uses a simple two-group design, to exemplify the issue of reliability, in examining the question of whether the cartoon SpongeBob SquarePants has more inappropriate content than does the cartoon Caillou.

Reliability- Inter-rater Reliability in Psychology Experiments

心理学实验的可靠性

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In order to study something scientifically, a ...

Placebos in Research

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

Clinical research focuses on the efficacy of treatments for addressing disorders and illnesses. A challenge with this type of research is that participants often have pre-existing beliefs about the treatment, particularly expectations that the treatment will work.
Though it has been practiced around the world for centuries, yoga is a relatively recent fitness craze in the United States with a wide range of alleged benefits, including the belief that it improves one&#8217;s creativity. However, it is not always clear whether yoga is actually creating the benefits, like improved creativity, or the yoga practitioner&#8217;s expectations are really the cause.
This video demonstrates a two-group design that examines whether a person who believes he or she is doing yoga (but in reality is not) experiences similar benefits to a person who actually does yoga. Specifically, this study looks at whether there is a placebo effect such that merely believing you are doing yoga benefits creativity.&#160;
Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented and the margin of error accompanied by studying human behavior is sufficiently accounted for. Further, human participants for research like this are often readily available and the experiment is quick and inexpensive to replicate. In this video we demonstrate this experiment using just one participant. However, as represented in the results, we used a total of 80 (40 for each condition) participants to reach the experiment&#8217;s conclusions.

Designing Experiment to Determine the Extent of Placebo Effects

安慰剂的研究

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

Clinical research focuses on the efficacy of ...

Manipulating an Independent Variable through Embodiment

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In any experiment, the researcher attempts to manipulate participants in one group to have different thoughts, experiences, or feelings than the other groups in the study.&#160; Some manipulations are overt, while others can be quite subtle. Embodiment is a growing research area focused on the theory that subtle physical experiences can unconsciously influence a person&#8217;s thoughts. For example if a person physically smiles, it often leads to elevated mood. That is, the physical experience of smiling changes the way a person feels.
This video uses a two-group experiment to see if the physical sensation of weight will lead people to be stricter by giving harsher forms of discipline to fellow students who violated campus policies.&#160;

操纵一个独立的变量，通过体现

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

In any experiment, the researcher attempts to ...

Experimentation using a Confederate

Source: Laboratories of <a href="https://www.jove.com/author/Gary_Lewandowski">Gary Lewandowski</a>, Dave Strohmetz, and Natalie Ciarocco&#8212;Monmouth University

When orchestrating an experiment, it is important that the experience elicits the most natural reactions from the participants as possible. Researchers accomplish much of this through their creation of the experimental settings.
Many research projects focus on interactions between two or more people.&#160; In these situations the environment or setting must often be less natural; often only one person can be a true participant and others in the study need to be &#8220;confederates,&#8221; that is, allegedly unbiased participants whom, in actuality, act according to the researcher&#8217;s directions.
This video uses a two-group experiment to see if participants are more likely to imitate a person with more power versus similar power compared to the participant.&#160; The video also highlights the use of research confederates.
Psychological studies often use higher sample sizes than studies in other sciences.&#160; A large number of participants helps to better ensure that the population under study is better represented, i.e. the margin of error accompanied by studying human behavior is sufficiently accounted for.&#160; Further, human participants for research like this are often readily available and the experiment is quick and inexpensive to replicate so we want to use as many participants as possible.&#160; In this video we demonstrate this experiment using just one participant.&#160; However, as represented in the results, we used a total of 156 participants to reach the experiment&#8217;s conclusions.

Education Experimental Psychology

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Education Experimental Psychology

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