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Research

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

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

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

2D NMR: Overview of Heteronuclear Correlation Techniques

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

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

2D NMR: Homonuclear Correlation Spectroscopy (COSY)

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

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

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)

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

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

Two-Dimensional (2D) NMR: Overview

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

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

2D NMR: Overview of Homonuclear Correlation Techniques

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

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

Double Resonance Techniques: Overview

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

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

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

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

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

Nuclear Overhauser Enhancement (NOE)

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

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

&sup1;H NMR of Labile Protons: Temporal Resolution

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

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

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

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

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

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

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

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

Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation

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

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

Chemical Ionization (CI) Mass Spectrometry

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

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

Mass Spectrometry: Alkyl Halide Fragmentation

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

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

Electrospray Ionization (ESI) Mass Spectrometry

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

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

Matrix-Assisted Laser Desorption Ionization (MALDI)

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

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

Mass Spectrometry: Aldehyde and Ketone Fragmentation

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

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

Mass Spectrometry: Alcohol Fragmentation

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

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

Mass Spectrometry: Alkyne Fragmentation

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

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

Mass Spectrometry: Aromatic Compound Fragmentation

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

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

Mass Spectrometry: Cycloalkene Fragmentation

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

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

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

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

The reaction proceeds via the cleavage of two ...

Mass Spectrometry: Amine Fragmentation

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

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

Mass Spectrometry: Long-Chain Alkane Fragmentation

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

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

Color Afterimages

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Human color vision is impressive. People with normal color vision can tell apart millions of individual hues. Most amazingly, this ability is achieved with fairly simple hardware.
Part of the power of human color vision comes from a clever bit of engineering in the human brain. There, color perception relies on what is known as an &#39;opponent system.&#39; This means that the presence of one kind of stimulus is treated as evidence for the absence of another, and vice versa; absence of one kind of stimulus is taken as evidence for the presence of the other. In particular, in the human brain there are cells that fire both when they receive signals to suggest that blue light is present, or when they do not receive signals suggesting yellow light. Similarly, there are cells that fire in the presence of yellow or the absence of blue. Blue and yellow are thus treated as opponent values in one dimension, and can be thought of as negative versus positive values on one axis of a Cartesian plane. If a stimulus is characterized as having a negative value on that axis, it can&#39;t also have a positive value. So, if it is characterized as yellow, it can&#39;t also be characterized as blue. Similarly, green and red (or really, magenta), occupy another opponent dimension. There are cells in the human brain that respond to the presence of one or the absence of the other. Figures 1 and 2 explain color opponency in Cartesian terms.
<img alt="Figure 1" src="/files/ftp_upload/10194/10194fig1.jpg" /> 
Figure 1. Opponent color dimensions. The human brain processes color using an opponent dimensions system. This is a two-dimensional plane with blue and yellow occupying one axis, which can be thought of as simply positive or negative, and red and green occupying the other axis. The consequence of the system is that the brain processes the presence of some colors as indicating the absence of others, and vice-versa. All perceivable colors occupy a point in the opponent space.
<img alt="Figure 2" src="/files/ftp_upload/10194/10194fig2.jpg" /> 
Figure 2. All perceivable colors occupy a point in the opponent space. Shown here are examples of colors that have nonzero values in each of two dimensions of the opponent space.
One way that color opponency was discovered-in 1878 by Ewald Hering, even before scientists had access to techniques for imaging the brain itself-is through an illusion known as a color afterimage. Afterimages are still used today both to demonstrate the opponent properties of human color perception and to study them.
This video demonstrates how to create a color afterimage illusion, and a simple way to collect subjective perceptual responses from human observers.

Studying Color Perception Using Color Afterimages Visual Illusion

Reflejo de color

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Human color vision is impressive. People with normal color vision can tell apart ...

Finding Your Blind Spot and Perceptual Filling-in

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
In the back of everyone's eye is a small piece of neural tissue called the retina. The retina has photosensitive cells that respond to stimulation by light. The responses of these cells are sent into the brain through the optic nerve, a bundle of neural fibers. In each retina there is a place somewhere in the periphery where the outputs from retinal cells collect and the bundled optic nerve exits to the brain. At that location, there is no photosensitivity-whatever light reflects from the world and lands in that position does not produce a signal in the brain. As a result, humans have a blind spot, a place in the visual field for which they don't process incoming stimuli. 
However, people are not aware that they have blind spots; there is not an empty hole in the visual images in front of the eyes. So what do people see in their blind spots? The brain actually fills-in missing input based on the surroundings. 
This video demonstrates how to find a person's blind spot, and how to investigate the mechanisms of perceptual filling-in.

Encontrar un punto ciego y rellenarlo perceptualmente?

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
In the back of everyone's eye is a small piece of neural tissue called the retina. ...

Perspectives on Sensation and Perception

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
The study of sensation&#8212;how signals are transduced from sensory organs, like the eyes&#8212;and perception&#8212;how the brain interprets these messages&#8212;has a rich history dating back to the 19th century, when great strides were made in understanding the properties of light and how they relate to the visual system. Importantly, such sensory and perceptual processes determine what we see, feel, taste, and hear in our surroundings. However, many teaching methods don&#8217;t expose students to the very sensory events they&#8217;re trying to learn about. 
JoVE&#8217;s collection in Sensation and Perception fills this gap by showcasing visual and auditory illusions that viewers can actually experience for themselves, and delves into the their anatomical bases. For example, by watching the video "Color Afterimages," an observer will encounter the phenomenon of perceiving a blank star as occupied by a color, and learn how specific neurons are responsible for this effect. By emphasizing such perceptual tricks, this collection explores the assumptions that the brain uses to interpret information and create our perception of a complex world. 
The JoVE videos in Sensation and Perception provide an engaging introduction to this field in psychology. By letting viewers sit in the seat of a participant and take part in actual experiments, this collection emphasizes to students that their own brains can be deceived into perceiving something that isn&#8217;t there, as well as even removing objects from perception.

Perspectivas sobre sensación y percepción

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
The study of sensation&#8212;how signals are transduced from sensory organs, like ...

Motion-induced Blindness

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
One thing becomes very salient after basic exposure to the science of visual perception and sensation: what people see is a creation of the brain. As a result people may fail to see things, see things that are not there, or see things in a distorted way. 
To distinguish between physical reality and what people perceive, scientists use the term awareness to refer to what people perceive. To study awareness, vision scientists often rely on illusions-misperceptions that can reveal the ways that the brain constructs experience. In 2001, a group of researchers discovered a striking new illusion called motion-induced blindness that has become a powerful tool in the study of visual awareness.1
This video demonstrates typical stimuli and methods used to study awareness with motion-induced blindness.

Studying Visual Awareness and Motion-Induced Blindness

Ceguera inducida por movimiento

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
One thing becomes very salient after basic exposure to the science of visual ...

The Rubber Hand Illusion

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Reaching for objects, walking without hitting obstacles, landing on a chair as you sit (instead of falling to the floor), these and all our physical actions depend on an ability to perceive our own bodies in space, to know where our limbs are relative to one another and relative to the rest of the world. One way that the human brain encodes this information is called proprioception, the brain relies on its own control and feedback signals to keep track of limbs. Along with proprioceptive inputs, the human brain incorporates vision, touch, and even sound in order to represent the parts of the body in space. How does it combine all this information? In 1998, Botvinick and Cohen described a striking illusion, called the Rubber Hand Illusion, that has been used to investigate how the human brain integrates sensory and proprioceptive inputs to represent the body in space.1 This video will demonstrate how to induce the Rubber Hand Illusion and it will describe how it has been used by subsequent studies.

Studying Proprioception Using the Rubber Hand Illusion

La ilusión de la mano de goma

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Reaching for objects, walking without hitting obstacles, landing on a chair as you ...

The Ames Room

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
The most difficult challenge of visual perception is often described as one of recovering information about three-dimensional space from two-dimensional retinas. The retina is the light-sensitive tissue inside the human eye. Light is reflected from objects in the world, casting projections on the retina that stimulate these light sensitive cells. Objects that are side-by-side in the world will produce side-by-side stimulations on the retina. But objects that are more distant from the observer cannot produce more distant stimulations, compared to nearby objects that is. Distance-the third dimension-is collapsed on the retina.

So how do we see in three dimensions? The answer is that the human brain applies a variety of assumptions and heuristics in order to make inferences about distances given the inputs received on the retina. In the study of perception, there is a long tradition of using visual illusions as a way to identify some of these heuristics and assumptions. If researchers know what tricks the brain is using, they should be able to trick the brain into seeing things inaccurately. This video will show you how to build an Ames Room, a visual illusion that illustrates one of the assumptions applied by the human visual system in order to recover visual depth.

Ames Room Illusion: Perception of Size, Distance and Depth

La habitación de Ames

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
The most difficult challenge of visual perception is often described as one of ...

Inattentional Blindness

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
We generally think that we see things pretty well if they are close by and right in front of us. But do we? We know that visual attention is a property of the human brain that controls what parts of the visual world we process, and how effectively. Limited attention means that we can't process everything at once, it turns out, even things that might be right in front of us. 
In the 1960s, the renowned cognitive psychologist Ulrich Neisser began to demonstrate experimentally that people can be blind to objects that are right in front of them, literally, if attention is otherwise distracted. In the 1980s and 1990s, Arien Mack and Irvin Rock followed up on Neisser's work, developing a simple paradigm for examining how, when, and why distracted attention can make people fail to see the whole object. Their experiments, and Neisser's, did not involve people with brain damage, disease, or anything of the sort, just regular people who failed to see objects that were right in front of them. This phenomenon has been called inattentional blindness. This video will demonstrate basic procedures for investigating inattentional blindness using the methods of Mack and Rock.1

Investigating Visual Awareness and Inattentional Blindness

Ceguera por falta de atención

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
We generally think that we see things pretty well if they are close by and right in ...

Spatial Cueing

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Attention refers to the limited human ability to select some information for processing at the expense of other stimuli in the environment. Attention operates in all sensory modalities: vision, hearing, touch, even taste and smell. It is most often studied in the visual domain though. A common way to study visual attention is with a spatial cueing paradigm. This paradigm allows researchers to measure the consequences of focusing visual attention in some locations and not others. This paradigm was developed by psychologist Michael Posner in the late 70s and early 80s in a series of papers in which he likened attention to a spotlight, selectively illuminating some portion of a scene.1,2 This video demonstrates standard procedures for a spatial cueing experiment to investigate visual attention.

Spatial Cueing Task to Investigate Covert Visual Attention

Localización espacial

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Attention refers to the limited human ability to select some information for ...

The Attentional Blink

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
In order for recognition of a certain stimulus to take place, visual attention needs to be directed towards said stimulus. To the earliest parts of the visual system, objects are not objects, they are collections of visual features-lines, corners, changes in texture, color, and light. Attention is the resource that is necessary for later processing in order to recognize what a given bundle of features adds up to. This makes attention a central focus of research. One especially important set of questions concerns how people sustain attention, that is, the extent to which they can continuously maintain a focus of attention from moment-to-moment. It is now known that sustained attention takes great effort. When attention needs to be focused very rapidly on something that is moving or changing very quickly, the effort involved causes a momentary lapse in attention once it is disengaged. This kind of lapse in attention is called an attentional blink. It is like the brain blinks for a moment, shutting down attention for a rest. Stimuli that appear during an attentional blink will not be perceived. 
In 1992, a group of researchers devised a paradigm to study the attentional blink, and the paradigm has come to be known by the same name.1 It demonstrates some of the challenges to maintaining focused attention. This video demonstrates how to implement the attentional blink paradigm in order to study sustained visual attention.

Sustained Visual Attention and the Attentional Blink Paradigm

Parpadeo atencional

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
In order for recognition of a certain stimulus to take place, visual attention ...

Crowding

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Human vision depends on light-sensitive neurons that are arranged in the back of the eye on a tissue called the retina. The neurons, called the rods and cones because of their shapes, are not uniformly distributed on the retina. Instead, there is a region in the center of the retina called the macula where cones are densely packed, and especially so in a central sub-region of the macula called the fovea. Outside the fovea there are virtually no cones, and rod density decreases considerably with greater distance from the fovea. Figure 1 schematizes this arrangement. This kind of arrangement is also replicated in the visual cortex: Many more cells represent stimulation at the fovea compared to the periphery.
<img alt="Figure 1" src="/files/ftp_upload/10280/10280fig1.jpg" /> 
Figure 1. Schematic depiction of the human eye and the distribution of light-sensitive receptor cells on the retina. The pupil is the opening in the front of the eye that allows light to enter. Light is then focused onto the retina, a neural tissue in the back of the eye that is made of rods and cones, light-sensitive cells. At the center of the retina is the macula, and in the center of the macula is the fovea. The graph schematizes the density of rod and cone receptors on the retina as a function of their position. Cones, which are responsible for color vision, are found almost exclusively in the fovea. Rods, which support seeing in low-light conditions, are similarly clustered more heavily near the fovea, with quickly falling densities outside the macula.
The result: We see very well in the part of space that our eyes are pointing at directly, the part of space stimulating the fovea; but we actually don&#39;t see very well in the periphery. We don&#39;t really notice it though, because our eyes move around constantly, building a representation of space from many individual fixations.
One way to study the properties of peripheral vision is with a phenomenon known as crowding.1 Crowding refers to an inability to recognize objects in clutter, and we experience crowding especially strongly when objects are shown in the periphery. Figure 2a is an example in which you should be able to experience crowding: Look at the cross in the center, and see if you can report the letter that is in the middle of the pack on the right. It is probably pretty hard. Now in Figure 2b try to report the letter on the left. Much easier! In this figure the letter is not crowded and there is no clutter around it, so it is easier to recognize.
<img alt="Figure 2a" src="/files/ftp_upload/10280/10280fig2a.jpg" />
Figure 2a. Crowded stimulus. Fixate the cross in the center, and see if you can recognize the letter in the middle of the pack on the left. It should be difficult, because the letters are in the periphery, and the central letter is crowded by the letters around it.
<img alt="Figure 2b" src="/files/ftp_upload/10280/10280fig2b.jpg" />
Figure 2b. Uncrowded stimulus. This stimulus is identical to Figure 2a, except that the letter G is uncrowded-no other letters surround it. Even while fixating the cross, the letter should be easy to recognize, even though it is just as much in the periphery as the G in Figure 2a.
This video will demonstrate how to design and implement a crowding experiment with letters as stimuli.

Investigating Crowding in Peripheral Vision

Hacinamiento?

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Human vision depends on light-sensitive neurons that are arranged in the back of ...

The Inverted-face Effect

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
In perception, it is often the case that the ability to recognize and interpret complex stimuli feels effortless but actually demands complicated and intensive processing. This is because processing is specialized and automated for certain types of very important stimuli. Among the best examples of this phenomenon is face processing. People do not try to detect and recognize faces. It just seems to happen. However, detecting faces and telling them apart from one another is actually a demanding computational task.

Human facial recognition abilities rely on specialized computations and dedicated brain networks. One simple demonstration of this is the inverted-face effect. Recognizing upside-down faces is far more difficult than recognizing them right-side up, but the same is not true for many other kinds of visual objects. The inverted-face effect is demonstrated in a variety of ways. This video shows an incidental encoding memory paradigm for investigating facial processing and the inverted-face effect.

The Inverted-face Effect: Face Recognition Using Incidental Encoding Memory

El efecto de cara invertida

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
In perception, it is often the case that the ability to recognize and interpret ...

The McGurk Effect

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Spoken language, a singular human achievement, relies heavily on specialized perceptual mechanisms. One important feature of language perception mechanisms is that they simultaneously rely on auditory and visual information. This makes sense, because until modern times, a person could expect that most language would be heard in face-to-face interactions. And because producing specific speech sounds requires precise articulation, the mouth can supply good visual information about what someone is saying. In fact, with an up-close and unobstructed view of someone&#39;s face, the mouth can often supply better visual signals than speech supplies auditory signals. The result is that the human brain favors visual input, and uses it to disambiguate inherent ambiguity in spoken language.
This reliance on visual input to interpret sound was described by Harry McGurk and John Macdonald in a paper in 1976 called Hearing lips and seeing voices.1 In that paper, they described an illusion that arises through a mismatch between a sound recording and a video recording. That illusion has become known as the McGurk effect. This video will demonstrate how to produce and interpret the McGurk effect.

The McGurk Effect: Audiovisual Illusion and Speech Perception

El efecto McGurk

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Spoken language, a singular human achievement, relies heavily on specialized ...

Just-noticeable Differences

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Psychophysics is a branch of psychology and neuroscience that tries to explain how physical quantities are translated into neural firing and mental representations of magnitude. One set of questions in this area pertains to just-noticeable differences (JND): How much does something need to change in order for the change to be perceivable? To pump intuitions about this, consider the fact that small children grow at an enormous rate, relatively speaking, but one rarely notices growth taking place on a daily basis. However, when the child returns from sleep-away camp or when a grandparent sees the child after a prolonged absence, just a few weeks of growing is more than perceptible. It can seem enormous! Changes in height are only noticed after an absence because the small changes that take place on a day-to-day basis are too small to be perceivable. But after an absence, many small changes add up. So how much growth needs to take place to be noticeable? The minimal amount is the JND.
Psychologists and neuroscientists measure JND in many domains. How much brighter does a light need to be to be noticed? How much louder does a sound need to be? They often obtain the measurements by employing a forced-choice paradigm. This video will focus on size, demonstrating a standard approach for measuring a JND when the area of a shape changes.

Measuring Just-noticeable Differences Using Shape Size Changes

Diferencias apenas perceptibles

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Psychophysics is a branch of psychology and neuroscience that tries to explain how ...

The Staircase Procedure for Finding a Perceptual Threshold

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Psychophysics is the name for a set of methods in perceptual psychology designed in order to relate the actual intensity of stimuli to their perceptual intensity. One important aspect of psychophysics involves the measurement of perceptual thresholds: How bright does a light need to be for a person to be able to detect it? How little pressure applied to the skin is detectable? How soft can a sound be and still be heard? Put another way, what are the smallest amounts of stimulation that humans can sense? The staircase procedure is an efficient technique for identifying a person&#39;s perceptual threshold.

This video will demonstrate standard methods for applying the staircase procedure in order to identify a person&#39;s auditory threshold, that is, the minimal volume necessary for a tone to be perceived.

El procedimiento de la escalera para encontrar un umbral Perceptual

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Psychophysics is the name for a set of methods in perceptual psychology designed in ...

Object Substitution Masking

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Visual masking is a term used by perceptual scientists to refer to a wide range of phenomena in which in an image is presented but not perceived by an observer because of the presentation of a second image. There are several different kinds of masking, many of them relatively intuitive and unsurprising. But one surprising and important type of masking is called Object Substitution Masking. It has been a focus of research in vision science since it was discovered, relatively recently, around 1997 by Enns and Di Lollo.1
This video will demonstrate standard procedures for how to conduct an object substitution experiment, how to analyze the results, and it will also explain the hypothesized causes for this unusual form of masking.

Object Substitution Masking and Conscious Perception of Shapes

Enmascaramiento de sustitución del objeto

Source: Laboratory of Jonathan Flombaum&#8212;Johns Hopkins University
Visual masking is a term used by perceptual scientists to refer to a wide range of ...

Education Sensation And Perception

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Education Sensation And Perception

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