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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: Amine Fragmentation

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

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

Mass Spectrometry: Cycloalkene Fragmentation

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

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

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

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

The reaction proceeds via the cleavage of two ...

Mass Spectrometry: 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: 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 ...

Determining Spatial Orientation of Rock Layers with the Brunton Compass

Source: Laboratory of Alan Lester - University of Colorado Boulder
Most rock units exhibit some form of planar surfaces or linear features. Examples include bedding-, fault-, fracture-, and joint-surfaces, and various forms of foliation and mineral alignment. The spatial orientation of these features form the critical raw data used to constrain models addressing the origin and subsequent deformation of rock units.
Although now over 100 years since its invention and introduction, the Brunton compass (Figure 1) remains a central tool in the modern geologist&#8217;s arsenal of field equipment. It is still the primary tool used to generate field data regarding the geometric orientation of planar rock surfaces or linear rock features. These orientation measurements are referred to as strike and dip, and provide the fundamental data for making geologic maps. Furthermore, the Brunton Compass can also function as a traditional compass for location exercises and triangulation. Finally, it can also serve as a pocket transit for measuring angular elevations.
<img alt="Figure 1" src="/files/ftp_upload/10086/10086fig1.jpg" /> 
Figure 1. The Brunton compass.

Brunton Compass: Strike and Dip Measurement of Rock Layers

Bestimmung der räumlichen Orientierung von Gesteinsschichten mit dem Brunton-Kompass

Source: Laboratory of Alan Lester - University of Colorado Boulder
Most rock units exhibit some form of planar surfaces or linear features. Examples ...

Using Topographic Maps to Generate Topographic Profiles

Source: Laboratory of <a href="https://www.jove.com/author/Alan_Lester">Alan Lester</a> - University of Colorado Boulder
Topographic maps are &#34;plan-view&#34; representations of Earth&#39;s three-dimensional surface. They are a standard type of map-view that provides an overhead, or aerial, perspective.
Among the defining features of a topographic map are the contour lines that indicate locations of constant elevation. The elevation interval between the contour lines is dependent on the level of detail provided by the map and the kind of topography present. For example, regions with significant topographic variation might require contour lines separated by 40-100 ft., whereas generally flat-lying regions with little topographic variation might have more broadly separated 10-20 ft. contours.
To an experienced user of such maps, the patterns made by the topographic lines are representative of various landform patterns, such as ridges, valleys, hills, and plateaus.

Verwendung von Topografischen Karten zur Erstellung von topografischen Profilen

Source: Laboratory of Alan Lester - University of Colorado Boulder
Topographic maps are &#34;plan-view&#34; representations of Earth&#39;s ...

Making a Geologic Cross Section

Source: Laboratory of <a href="https://www.jove.com/author/Alan_Lester">Alan Lester</a> - University of Colorado Boulder
Geologic maps were first made and utilized in Europe, in the mid-to-late 18th century. Ever since, they have been an important part of geological investigations all around the world that strive to understand rock distributions on the surface of the earth, in the subsurface, and their modification through time. A modern geologic map is a data-rich representation of rocks and rock-structures in a two-dimensional plan view. The base for most geologic maps is a topographic map, onto which color variations have been placed to represent specific rock units. The boundaries between the rock units are called contacts. In addition to the contact lines, geologic maps contain symbols that represent key features, such as the dip and strike of the rock units, anticlines and synclines, and the traces of fault surfaces.
Although the two-dimensional map-view is useful, one of a geologist&#39;s key tasks is to infer the type and orientation of rocks in the subsurface. This is done using geologic rules, inferences, and projections downward from the surface. The result is a geologic cross section, a view that essentially provides a cutaway image, much like one would see on a canyon wall or in a roadcut.
This hypothetical slice into the earth, providing a third dimension (depth), is the key to a host of geological applications. Cross sections are used to assess temporal models of rock formation through time. In other words, the goal is to recreate a step-by-step sequence of which rocks and structures came first, last, and in between. They&#39;re also used to determine specific modes of deformation - whether rocks have undergone compressional, extensional, or other stresses.
Geologic cross sections help to identify regions of groundwater movement, evaluate potential sites for economic mineral deposits, and locate oil and gas reservoirs.

Erstellen eines geologischen Querschnitts

Source: Laboratory of Alan Lester - University of Colorado Boulder
Geologic maps were first made and utilized in Europe, in the mid-to-late 18th century. ...

Physical Properties Of Minerals I: Crystals and Cleavage

Source: Laboratory of <a href="https://www.jove.com/author/Alan_Lester">Alan Lester</a> - University of Colorado Boulder
The physical properties of minerals comprise various measurable and discernible attributes, including color, streak, magnetic properties, hardness, crystal growth form, and crystal cleavage. Each of these properties are mineral-specific, and they are fundamentally related to a particular mineral&#8217;s chemical make-up and atomic structure.
This experiment examines two properties that stem primarily from symmetric repetition of fundamental, structural atomic groupings, called unit cells, within a crystal lattice, a crystal growth form, and crystal cleavage.
Crystal growth form is the macroscopic expression of atomic-level symmetry, generated by the natural growth process of adding unit cells (the molecular building blocks of minerals) to a growing crystal lattice. Zones of rapid unit-cell-addition become the edges between the planar surfaces, i.e. faces, of the crystal.
It is important to recognize that rocks are aggregates of mineral grains. Most rocks are polymineralic (multiple kinds of mineral grains) but some are effectively monomineralic (composed of a single mineral). Because rocks are combinations of minerals, rocks are not referred to as having crystal form. In some cases, geologists refer to rocks as having a general cleavage, but here the term is simply used to refer to repetitive breaking surfaces and is not a reflection of atomic crystal structure. So, in general, the terms crystal form and crystal cleavage are used in reference to mineral samples and not rock samples.

Mineral Crystals: Unit Cells, Lattices and Cleavage Planes

Physikalische Eigenschaften von Mineralien I: Kristalle und Spaltbarkeit

Source: Laboratory of Alan Lester - University of Colorado Boulder
The physical properties of minerals comprise various measurable and discernible ...

Physical Properties Of Minerals II: Polymineralic Analysis

Source: Laboratory of <a href="https://www.jove.com/author/Alan_Lester">Alan Lester</a> - University of Colorado Boulder
The physical properties of minerals include various measurable and discernible attributes, including color, streak, magnetic properties, hardness, crystal growth form, and crystal cleavage. These properties are mineral-specific, and they are fundamentally related to a particular mineral&#8217;s chemical make-up and atomic structure.
This video examines several physical properties that are useful in field and hand sample mineral identification&#8212; color, luster, streak, hardness, magnetism, and reaction with acid. Unlike crystal form and crystal cleavage, these properties are somewhat more closely linked to mineral chemical composition than to atomic structure, but both do play a role.
It is important to recognize that rocks are aggregates of mineral grains. Most rocks are polymineralic (multiple kinds of mineral grains) but some are effectively monomineralic (composed of a single mineral). Unlike crystal form and cleavage, which are terms reserved for mineral specimens, geologists might on occasion refer to a rock as having a general sort of color, hardness, magnetism, or reaction with acid. In other words, the physical properties looked at here are potentially appropriate for use with rocks as well as with specific minerals.

Physical Properties of Minerals and Mineral Characterization

Physikalische Eigenschaften von Mineralien II: Polymineralische Analyse

Source: Laboratory of Alan Lester - University of Colorado Boulder
The physical properties of minerals include various measurable and discernible ...

Igneous Volcanic Rock

Source: Laboratory of <a href="https://www.jove.com/author/Alan_Lester">Alan Lester</a> - University of Colorado Boulder
Igneous rocks are the products of cooling and crystallization of magma. Volcanic rocks are a particular variety of igneous rock, forming as a consequence of magma breaching the surface, then cooling and crystallizing in the subaerial environment.&#160;
Magma is liquid rock that typically ranges in temperature from approximately 800 &#176;C to 1,200 &#176;C (Figure 1). Magma itself is produced within the Earth via three primary melting mechanisms, namely the addition of heat, addition of volatiles, and decompression. Each mode of melt generation tends to produce specific types of magma and, therefore, distinct eruptive styles and structures.
<img alt="Figure 1" src="/files/ftp_upload/10037/10037fig1.jpg" /> 
Figure 1.&#160;Fresh lava breakout on Kilauea, Hawaii. Lava is the term for magma that is on Earth&#8217;s surface.

Igneous Volcanic Rock Formation: Magma Content, Viscosity and Explosivity

Magmatisches Eruptivgestein

Source: Laboratory of Alan Lester - University of Colorado Boulder
Igneous rocks are the products of cooling and crystallization of magma. Volcanic rocks ...

Igneous Intrusive Rock

Source: Laboratory of <a href="https://www.jove.com/author/Alan_Lester">Alan Lester</a> - University of Colorado Boulder
Igneous rocks are products of the cooling and crystallization of high temperature liquid rock, called magma. Magmatic temperatures typically range from approximately 800 &#176;C to 1,200 &#176;C. Molten rock is, perhaps luckily for humans, an anomaly on planet Earth. If a random and imaginary drill hole were made in the Earth, it would most likely not reach a region of truly and totally molten material until the outer core, at nearly 2,900 km beneath the surface (Earth&#39;s radius is 6,370 km). Even there, this molten material would predominantly consist of liquid iron, not true silicate rock, and be incapable of ever reaching Earth&#39;s surface.
Volcanic eruptions and igneous rocks do occur though, and they are evidence that there are indeed isolated regions of melting and magma generation within the Earth.

Igneous Intrusive Rock: Formation, Texture and Composition

Magmatisches Intrusivgestein

Source: Laboratory of Alan Lester - University of Colorado Boulder
Igneous rocks are products of the cooling and crystallization of high temperature ...

An Overview of bGDGT Biomarker Analysis for Paleoclimatology

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
Throughout this series of videos, natural samples were extracted and purified in search of organic compounds, called biomarkers, that can relate information on climates and environments of the past. One of the samples analyzed was sediment. Sediments accumulate over geologic time in basins, depressions in the Earth into which sediment flows through the action of fluid (water or air), movement, and gravity. Two main types of basins exist, marine (oceans and seas) and lacustrine (lakes). As one might guess, very different types of life live in these settings, driven in large part by the difference in salinity between them. Over the last few decades, organic geochemists discovered a toolbox of biomarker proxies, or compounds that can be used to describe climate or environment, some of which work in marine environments and some of which work in lacustrine. We turn our attention here to the lacustrine realm and branched glycerol dialkyl glycerol tetraethers (Figure 1).
In this section we focus on analysis of terrestrial paleotemperature using branched glycerol dialkyl glycerol tetrathers (Figure 1; brGDGTs) and the MBT/CBT proxy. This proxy was initially described by Weijers et al.1&#160;and is based on the distribution of ring and branch structures in brGDGTs. They found that the cyclization of branched tetraethers (CBT) was directly related to soil pH.
CBT = -log ((Ib + IIb) / (I + II))
And that the methylation of branched tetraethers (MBT) was determined by mean annual air temperature (MAAT) and, to a lesser extent, soil pH.
MBT = (I + Ib + Ic) / (I + Ib + Ic) + (II + IIb + IIc) + (III + IIIb + IIIc)
Thus, taken together and calibrated, MBT/CBT relates the distribution of brGDGTs to both soil temperature and pH.
MBT = 0.122 + (0.187 x CBT) + (0.020 x MAAT)
Branched GDGTs are thought to be membrane spanning lipids and their production was initially attributed to anaerobic Acidobacteria bacteria living in soils and peat2-5, but subsequent work suggested they could also be produced in oxic and anoxic lake and marine water columns and sediments6-9. The hypothesis holds that Acidobacteria transform sites of methylation into cyclizations in response to lowering temperature in order to increase unsaturation (cyclization effectively removes two hydrogen atoms) and maintain membrane fluidity (by analogy, saturated fat (butter) is a solid a room temperature while unsaturated fat (olive oil) is a liquid), but branched GDGTs have not yet been identified as the main membrane lipids in Acidobacteria cultures. Thus their exact provenance is unknown.
Calibration of branched GDGTs to environmental variables (temperature, pH, salinity, precipitation, etc.) is a topic of widespread research. Organic geochemistry laboratories all over the world are involved in the task of developing both global1,10&#160;and regional11-13&#160;calibrations between branched GDGTs and (primarily) temperature. Thus, the equations given above are regularly being refined and perfected.
Branched GDGTs are usually extracted from lacustrine sediments, although coastal marine sediments have also been investigated. The extracts undergo a silica gel column to purify the GDGTs from other compounds which may not be LC amenable or that may co-elute with GDGTs chromatographically. The GDGTs come out in the polar fraction that elutes in methanol.
Once the total lipid extract is purified, the extracted and purified sample is run on a high performance liquid chromatograph coupled to a chemical ionization mass spectrometer. The relative concentration of the GDGTs is determined by obtaining the area under the curve for the selected mass ion (m/z; Figure 1) for each of the compounds on computer software designed for just this purpose (such as Agilent Chemstation). These areas are then put into the selected calibration equation in order to arrive at a paleotemperature determination.
<img alt="Figure 1" src="/files/ftp_upload/10256/10256fig1.jpg" /> 
Figure 1. Structures of the branched GDGTs used for calculating temperature via MBT/CBT proxy (used with permission of Dr. Isla Casta&#241;eda, who produced the image). <a href="https://www.jove.com/files/ftp_upload/10256/10256fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<div class="chapter_text" id="script_0">
Organic compounds called biomarkers can be used in Earth Science as paleothermometers to relate information on climates and environments of the past.
Living organisms produce these biomarkers, which provide us with information about the environment in which they lived. They can act as a proxy to tell us information about past events, like the Earth&#8217;s temperature millions of years ago.
Terrestrial paleotemperature can be analyzed using biomarkers found in sediment from fresh-water basins. One key class of these biomarkers are branched glycerol dialkyl glycerol tetraethers, or branched GDGTs.
This video will introduce the area of study, called paleoclimatology, which investigates past changes in fresh-water environments over hundreds of millions of year. This helps elucidate current and future climate and environmental changes.
</div>
<div class="chapter_text" id="script_1">
Sediments accumulate over geologic time, due to fluid movement and gravity, in sedimentary basins, or low areas in the Earth&#8217;s crust. Sedimentary basins include oceans, which collect marine sediment, or lakes, which collect lacustrine sediment. Marine and lacustrine basins contain different types of organisms, driven in large part by the difference in salinity between them. Thus, marine and lacustrine basins contain different biomarkers.
Branched GDGTs are thought to be membrane-spanning lipids of anaerobic acidobacteria. Research suggests that the producing organisms change membrane properties in response to changing temperature.
This change is caused by the transformation of methylated sites on the branched GDGT&#8217;s to cyclized sites at colder temperatures, thereby enhancing membrane fluidity. This change in structure can then be correlated to temperature through a proxy. Proxies are measureable physical phenomena that are correlated to immeasurable variable.
This proxy relates the number of methylations or MBT, and cyclizations, or CBT, in the biomarker to temperature. An experimentally-derived equation can relate MBT and CBT to the past Mean Annual Air Temperature.
</div>
<div class="chapter_text" id="script_2">
To study the relationship between branched GDGT biomarkers and soil temperature, lacustrine sediment must be collected, extracted by one of three techniques, purified, and analyzed.
To begin studying the relationship between branched GDGT biomarkers and soil temperature, the lipid molecules are first extracted from lacustrine sediments, using a variety of techniques. Extraction via sonication is the simplest and least expensive method of obtaining the total lipid extract, or TLE, from a sediment sample. For this, an ultrasonic bath is used to agitate the sample in a vial containing organic solvent. A mixture of methanol and dichloromethane is used to extract biomarkers with a wide range of polarities. Another extraction technique utilizes Soxhlet extraction. A Soxhlet extractor enables the reflux, or continuous cycling, of organic solvent from a round-bottom flask upward into a condenser, which is cooled by cold water and returned. The condensed solvent falls into a glass fiber thimble containing the sample. Once full, the chamber siphons the organic solvent back into the round-bottom flask, enabling continuous extraction over time.
This technique is helpful in the extraction of large sediment masses, and the preparation of large volumes of standards for instrument calibration. Finally, accelerated solvent extraction, or ASE, is a trademarked method of extraction that utilizes high temperature and pressure to increase the kinetics of the extraction process. The ASE instrument holds up to 24 individual samples, and allows for precise control of all parameters in the extraction process. Due to its speed and simplicity of use, ASE is commonly used as the standard method of solvent extraction.
</div>
<div class="chapter_text" id="script_3">
Once the lipid sample is extracted using one of these techniques, it is purified in preparation for analysis. Typically, silica gel column chromatography is used to purify the lipid sample based on its polarity. For this, a small glass column is loaded with a fine powder of silica, called a gel. The column is then saturated with an apolar solvent, typically hexane, and then the sample loaded on the top. The separation of the extract is based on the affinity of the target compound for either the solid phase or the solvent phase.
Polar compounds, in this case branched GDGT&#39;s, are more attracted to the polar silica than the apolar hexane. Thus, the apolar compounds, such as hydrocarbons, the mid-polar compounds, such as ketones and alcohols, and the highly polar compounds, will travel the column at different rates and in response to solvents of increasing polarity.
The eluents are then collected in separate fractions.
The purified GDGT&#39;s are then analyzed using high performance liquid chromatography coupled to a mass spectrometer, or LC-MS. LC-MS first separates the compounds, and then analyzes them based on their mass-to-charge ratio.
This enables the determination of the relative concentration of each type of GDGT using the area under the curve for the selected mass ion. MBT is calculated as the fraction of the group 1 molecules to the total.
CBT is then calculated as a negative log using molecules in groups 1 and 2. MBT and CBT are then plugged into an experimentally-derived equation, in order to arrive at a paleotemperature determination.
</div>
<div class="chapter_text" id="script_4">
The determination of paleotemperature using biomarker proxies is useful in a range of applications in earth science.
First, paleothermometry enables the determination of the Earth&#39;s temperature over long periods of time. Using various techniques, the temperature of Earth has been estimated as far back as 500 million years. This tells us the envelope of temperature within which different forms of life evolved and informs investigations of the effects of temperature on Earth&#39;s biosphere, hydrosphere, lithosphere, and atmosphere in the past, and by extension, the future.
More recent trends in the Earths temperature can also be quantified against records constructed using paleothermometry. The Earths surface temperature has increased by nearly 1 degree from 1850 to the present with an accentuated warming trend in the last two decades. To understand the anthropogenic impact on global climate, accurate paleoclimate records must be developed and used as context.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s Overview of Branched Glycerol Dialkyl Glycerol Tetraether Paleothermometry. You should now understand how the branched GDGT biomarkers are used, and the overall technique of extracting and purifying them. The following videos in this series will go into more detail about this complex process.
Thanks for watching!
</div>

Ein Überblick über die bGDGT-Biomarker-Analyse für die Paläoklimatologie

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
Throughout this series of videos, natural samples were extracted and ...

An Overview of Alkenone Biomarker Analysis for Paleothermometry

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
Throughout this series of videos, natural samples were extracted and purified in search of organic compounds, called biomarkers, that can relate information on climates and environments of the past. One of the samples analyzed was sediment. Sediments accumulate over geologic time in basins, depressions in the Earth into which sediment flows through the action of fluid (water or air), movement, and gravity. Two main types of basins exist, marine (oceans and seas) and lacustrine (lakes). As one might guess, very different types of life live in these settings, driven in large part by the difference in salinity between them. Over the last few decades, organic geochemists discovered a toolbox of biomarker proxies, or compounds that can be used to describe climate or environment, some of which work in marine environments and some of which work in lacustrine. We turn our attention here to the marine realm and alkenone paleothermometry using the Uk&#39;37 sea surface temperature proxy.
The most well-established and widely applied open-ocean biomarker sea surface temperature (SST) proxy is Uk&#39;37.
Uk&#39;37 = (C37:2) / (C37:2 + C37:3) (see Herbert1 for a review)
The index is based on the ratio of two polyunsaturated long-chain alkyl ketones, called alkenones, produced by some classes of haptophyte algae2,3. Culture4,5&#160;and core-top sediment6&#160;calibration studies led to the development of the Uk&#39;37 Index as a quantitative SST proxy. Amazingly, the culture-based calibration of Prahl et al.4:
Uk&#39;37 = 0.034(SST) + 0.039,
And the core-top calibration of M&#252;ller et al.6,
Uk&#39;37 = 0.033(SST) + 0.044,
are statistically identical.
Reconstructed Uk&#39;37 temperatures correlate best with mean annual SST for a variety of climate and haptophyte production regimes in the global ocean7. Alkenones are detected in marine sediment cores of early Eocene to modern age8, and in exposed outcrops of uplifted marine sediment9&#160;suggesting they are very stable over geologic time, and thus useful as a paleoclimate tool. Uk&#39;37 has been used to document paleo sea surface temperature changes at decadal10&#160;to orbital11,12&#160;timescales and are therefore very versatile.
In the open ocean, the coccolithophores Emiliania huxleyi and Gephyrocapsa oceanica are responsible for most alkenone production. It is not yet known why these haptophytes alter the unsaturation ratio of alkenones based on growth temperature. It was initially thought that alkenones were components of haptophyte cell walls and that their unsaturation was adjusted in order to keep the membrane fluid, much like saturated fats are solid at room temperature, while unsaturated fats are fluid. However, experiments aimed at this question found that instead of being associated with the cell membranes, alkenones were associated with energy storage structures inside the cell. Thus, their use inside the cell remains an open question.
Recently, alkenones have been found in lacustrine environments. However, their utility has so far been limited. Different alkenone producers than those in the marine realm dwell in lakes and thus the calibration between water temperature and unsaturation (Uk&#39;37) is different. Moreover, this calibration is different between lakes, making the creation of a &#39;global&#39; calibration unlikely. Unfortunately, the creation of local calibrations is expensive and time consuming and so the future for Uk&#39;37 in lakes is also currently limited.
Alkenones are usually extracted from marine sediments. Very often the same organisms that produce alkenones produce fatty acid methyl esters of those alkenones called alkenoates. These compounds co-elute with the alkenones on a gas chromatograph and complicate their quantification. Therefore, these extracts will often undergo a saponification to remove alkenoates. Because the saponification produces carboxylic acids that are not gas chromatograph amenable, a silica gel column must be performed after the saponification to remove the carboxylic acids from the extract. The alkenones come out in the middle polarity ketone fraction that elutes in dichloromethane while the acids are left on the column. Lastly, in extreme cases, such as in sediments acquired from highly polluted areas, like estuaries near industrial centers, a urea adduction may also be required to remove unknown compounds that coelute with the alkenones on the gas chromatograph.
Once the total lipid extract is purified, the extracted and purified sample is run on a gas chromatograph coupled to a flame ionizing detector. The relative concentration of the two alkenones is determined by obtaining the area under the curve for each of the compounds on computer software designed for just this purpose (such as Agilent Chemstation). These areas are then put into the Uk&#39;37 ratio equation shown above to get a Uk&#39;37 value that ranges between 0 and 1. These Uk&#39;37 values are then mapped to sea surface temperature value using a calibration such as those described above.
<div class="chapter_text" id="script_0">
Paleothermometry is the calculation of past temperatures by analysis of specific chemicals in natural samples, like those left behind by prehistoric algae.
Algae are a diverse group of organisms that have been abundant in Earth&#39;s oceans and lakes for millennia. Certain chemical compounds, which are deposited in sediment by ancient algae, act as biomarkers &#8211; organic compounds that can provide researchers with valuable insight into Earth&#8217;s history. In fact, analysis of algal biomarker content in sediment allows researchers to determine the Earth&#8217;s temperature hundreds of millions of years ago.
One such record comes from some species of coccolithophores. These algae produce varying amounts of alkenones, a class of robust biomarkers, based on the temperature of their environment. Alkenone analysis is primarily used to calculate the sea surface temperature of Earth&#39;s oceans eons and eons ago.
This video will illustrate the use of alkenones in paleoclimatology and describe the process of isolating, purifying, and analyzing alkenones to calculate past sea surface temperature.
</div>
<div class="chapter_text" id="script_1">
As its name implies, &#8220;Alkenone paleothermometry&#8221; is based on the analysis if lipids, known as alkenones Alkenone paleothermometry is based on alkenones; long-chain, unsaturated alkyl ketones that contain 37 carbon atoms and 2 to 4 double bonds. Each double bond is a site of unsaturation. At low sea surface temperatures, alkenone producers generate more unsaturated alkenones than saturated. The ratio of saturation to unsaturation is known as the Alkenone Unsaturation Index.
The alkenones usually evaluated are C37:2 and C37:3, which have 37 carbons and two or three double bonds, respectively. The Unsaturation Index of these alkenones, or the UK&#39;37, is positively related to sea surface temperature. The analytical method know as gas chromatography is generally sensitive enough to separate these alkenones from one another. However, alkenone-producing algae often also generate chemically-similar fatty acid methyl esters, or alkenoates, which cannot be distinguished from alkenones using this technique.&#160;Hydrocarbon contamination from pollution may also further muddy chromatographic analysis. To accurately determine relative alkenone concentration, alkenoates and unknown hydrocarbons must be removed before analysis by the methods of saponification and urea adduction.
Now that the relationship of sediment alkenone ratios to sea surface temperature has been reviewed, let&#39;s look at the techniques for their purification from a total lipid extract and analysis of the unsaturation ratio.
</div>
<div class="chapter_text" id="script_2">
Once marine sediment has been collected and extracted, the total lipid extract, or TLE, must go through a multistep purification process, and analyzed. First, the extract undergoes saponification to convert alkenoates into carboxylate salts and methanol using a strong base and heat. Other fatty acid esters present in the TLE will be saponified into salts and glycerol.
After cooling the mixture to room temperature, an aqueous salt solution is added to form salts and glycerol. The mixture is then acidified to protonate the carboxylate anions, producing fatty acids. Finally, the alkenones and fatty acids are extracted from the mixture with hexane.
Silica gel chromatography is then performed to remove both apolar compounds and the polar fatty acids produced by saponification. The dried and saponified TLE is dissolved in hexane and then loaded onto a column. Silica retains polar compounds more strongly than apolar ones.
First, apolar compounds are removed with an apolar solvent, like hexane. Next, alkenones are eluted by a moderately polar solvent, such as dichloromethane, leaving the highly polar fatty acids and other unwanted polar compounds on the column.
If the original sediment sample was collected from a highly polluted area, urea adduction is performed to remove any remaining highly branched or cyclic hydrocarbons. The dried mid-polarity fraction is dissolved in a solvent mixture in which the strongly polar urea is minimally soluble, such as DCM and hexane. A concentrated solution of urea in methanol is then added to the TLE, causing urea crystals to precipitate.
Straight-chain molecules such as alkenones fit into the spaces between molecules in the urea crystal lattice, but highly branched and cyclic molecules do not, and are expelled.
Once crystal growth has finished, the urea crystals are dried and then washed with an apolar solvent to remove expelled compounds. Then, the crystals are dissolved in a small amount of water. The alkenones are extracted from the water with an apolar solvent for analysis.
</div>
<div class="chapter_text" id="script_3">
While all previous purification steps did not differentiate between alkenone species, small differences in boiling point and molecular structure are sufficient for separation on a gas chromatography column. When paired with a flame-ionization detector, relative concentrations of the&#160;alkenones, can be determined.
Molecules are identified on the chromatogram by their retention time, or the time needed for the compound to be exit the column. The retention times of the desired compounds are ascertained with alkenone standards.
The relative concentrations of the alkenones are determined from analysis of the areas under the peaks of interest. The UK&#39;37 value is then calculated from the concentrations of C37:2and C37:3 in the sample. With the sea surface temperature proxy relationship and the UK&#39;37 value, the analyst can solve for sea surface temperature at the time of the sediment deposition.
</div>
<div class="chapter_text" id="script_4">
Many different facets of Earth&#39;s history can be investigated by analysis of sediment and sedimentary rock.
Biostratigraphy is the study of determining the ages of layers, or strata, of rock by analysis of the fossils present. As there are many sources of sediment, sedimentary rocks from the same time period may have dramatically different compositions around the world. Certain sets of species throughout Earth&#39;s history, such as the ammonites, existed worldwide and underwent rapid evolution. If visually dissimilar rock strata both contain the same species of ammonite, then a temporal correlation between the strata can be drawn. When combined with techniques such as paleothermometry, extensive information about Earth&#39;s history can be determined from fossil records in natural samples.
Many species of foraminifera, or forams, are found in marine sediments worldwide. Forams have calcium carbonate shells and have existed throughout Earth&#39;s oceans for millions of years. Many species&#160;live on the ocean floor, and thus can provide temperature information about deeper parts of the ocean. The magnesium to calcium ratio of forams&#160;corresponds to temperature, as they incorporate more magnesium into their shells in warmer climates. The multitude of species and the abundance of forams makes their fossil record useful for tracking changes in ocean currents throughout Earth&#39;s history and for biostratigraphy.
As tectonic plates diverge, new rock forms between them. Correspondingly, the properties of the rock surrounding a divergent plate boundary provide information about plate movements over time. For instance, changes in Earth&#39;s magnetic field are preserved in some minerals found in fossils, rock, and sediment. The discovery of symmetric changes in magnetism about mid-ocean ridges significantly contributed to the current understanding of seafloor spreading and plate tectonics.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s Overview of Alkenone Paleothermometry. You should now understand the principles of paleothermometry and the relationship of alkenone ratios in marine sediment to sea surface temperature. The following videos in this series will go into more detail about this complex process.
Thanks for watching!
</div>

Ein Überblick über die Alkenon-Biomarker-Analyse für die Paläothermometrie

Sonication Extraction of Lipid Biomarkers from Sediment

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The material comprising the living &#34;organic&#34; share of any ecosystem (leaves, fungi, bark, tissue; Figure 1) differs fundamentally from the material of the non-living &#34;inorganic&#34; share (rocks and their constituent minerals, oxygen, water, metals). Organic material contains carbon linked to a series of other carbon and hydrogen molecules (Figure 2), which distinguishes it from inorganic material. Carbon&#39;s wide valency range (-4 to +4) allows it to form up to four separate covalent bonds with neighboring atoms, usually C, H, O, N, S, and P. It can also share up to three covalent bonds with a single other atom, such as the triple bond in the often-poisonous cyanide, or nitrile, group. Over the past 4.6 billion years, this flexibility has led to an amazing array of chemical structures, which vary in size, complexity, polarity, shape, and function. The scientific field of organic geochemistry is concerned with the identification and characterization of the whole range of detectable organic compounds, called biomarkers, produced by life on this planet, as well as others, through geologic time.
<img alt="Figure 1" src="/files/ftp_upload/10055/10055fig1.jpg" /> 
Figure 1. Organic material, such as trees, leaves, and moss, are chemically and visually distinct from inorganic material, such as pavement.

Ultraschall-Extraktion von Lipid-Biomarkern aus Sediment

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The material comprising the living &#34;organic&#34; share of any ecosystem ...

Soxhlet Extraction of Lipid Biomarkers from Sediment

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
Every lab needs standards that track the performance, accuracy, and precision of its instruments over time to ensure a measurement made today is the same as a measurement made a year from now (Figure 1). Because standards must test the performance of instruments over a long period of time, large volumes of the standards are often required. Many chemical standards can be purchased from retail scientific companies, like Sigma-Aldrich and Fisher. However, some compounds that occur in nature and that are relevant to paleoclimatic studies have not yet been isolated and purified for purchase. Therefore, these compounds need to be extracted from natural samples, and because of the large volumes of standards required, large volumes of sediment need to be extracted. The Accelerated Solvent Extraction (Dionex) and sonication extractions are not appropriate for the extraction of such large sediment volumes. In these circumstances, a Soxhlet extraction is used.
<img alt="Figure 1" src="/files/ftp_upload/10096/10096fig1.jpg" /> 
Figure 1. Schematic depicting how chemical standard tracks the performance of an instrument through time. The dashed line represents a 1:1 relationship between the accepted and measured (on the instrument) value of a variable. Each star is a weekly measurement of the chemical standard. Green stars represent standards that are accurate. Red stars reflect those that are not accurate indicating that the instrument requires corrective maintenance.

Soxhlet-Extraktion von Lipid-Biomarkern aus Sediment

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
Every lab needs standards that track the performance, accuracy, and ...

Extraction of Biomarkers from Sediments - Accelerated Solvent Extraction

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The distribution of a group of organic biomarkers called glycerol-dialkyl glycerol-tetraethers (GDGTs), produced by a suite of archaea and bacteria, were found in modern sediments to change in a predictable manner in response to air or water temperature1,2. Therefore, the distribution of these biomarkers in a sequence of sediments of known age can be used to reconstruct the evolution of air and/or water temperature on decadal to millennial timescales (Figure 1). The production of long high-resolution records of past climates, called paleoclimatology, depends on the rapid analysis of hundreds, possibly thousands of samples. Older extraction techniques, such as sonication or Soxhlet, are too slow. However, the newer Accelerated Solvent Extraction technique was designed with efficiency in mind.
<img alt="Figure 1" src="/files/ftp_upload/10097/10097fig1.jpg" /> 
Figure 1. An example of a paleoclimate record showing changes in sea surface temperature (SST) in the eastern Mediterranean Sea during the past ~27,000 years3. This record comprises ~115 samples and is based on the isoprenoidal GDGT-based TEX86 SST proxy.

Accelerated Solvent Extraction of Total Lipid from Sediments

Extraktion von Biomarkern aus Sedimenten - beschleunigte Lösemittelextraktion

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The distribution of a group of organic biomarkers called glycerol-dialkyl ...

Conversion of Fatty Acid Methyl Esters by Saponification for Uk'37 Paleothermometry

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The product of an organic solvent extraction, a total lipid extract (TLE), is often a complex mixture of hundreds, if not thousands, of different compounds. The researcher is often only interested in a handful of compounds or, if interested in many, may need to remove unwanted constituents that are&#34;in the way&#34; or co-eluting. For example, the concentrations of individual compounds in a sample are often determined on a gas chromatograph coupled to a flame-ionizing detector (GC-FID), because the relationship between FID response (in pA) and the amount of compound in a sample (e.g., ng/&#181;L) is both linear and sensitive. The GC portion of the instrument separates different compounds in a sample based on their boiling point, chemical structure, and affinity with a solid phase that can change according to application. The result is a chromatogram (Figure 1), showing the separation of different chemical constituents in time, as well as their relative concentration (calculated as the area under the curve). However, sometimes more than one compound elutes off the GC at a time (Figure 1). In this case, sample purification is required before compounds can be confidently quantified. 
&#160;
<img alt="Figure 1" src="/files/ftp_upload/10158/10158fig1.jpg" /> 
Figure 1. A chromatogram showing the separation of different chemical constituents over time and their relative concentration (area under the curve). Co-eluting and separated peaks are shown.

Purification of Alkenones via Saponification for Uk&#039;37 Paleothermometry

Umwandlung von Fettsäuremethylestern durch Verseifung für die Uk'37-Paläothermometrie

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The product of an organic solvent extraction, a total lipid extract (TLE), ...

Purification of a Total Lipid Extract with Column Chromatography

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The product of an organic solvent extraction, a total lipid extract (TLE), is often a complex mixture of hundreds, if not thousands, of different compounds. The researcher is often only interested in a handful of compounds. The compounds of interest may belong to one of several classes of compounds, such as alkanes, ketones, alcohols, or acids (Figure 1), and it may be useful to remove the compound classes to which it does not belong in order to get a clearer view of the compounds you are interested in. For example, a TLE may contain 1,000 compounds, but the Uk&#39;37 sea surface temperature proxy is based on only two compounds (alkenones) and the TEX86 sea surface temperature proxy is based on only four (glycerol dialkyl glycerol tetraethers). It would behoove the researcher to remove as many of the compounds they are not interested in. This makes the instrumental analysis of the compounds of interest (alkenones or GDGTs) less likely to be complicated by other extraneous compounds.
In other cases, an upstream purification technique may have produced compounds you wish to now remove from the sample, such as the production of carboxylic acids during saponification in our previous video. In both of the above cases the purification technique called column chromatography is very useful.
<img alt="Figure 1" src="/files/ftp_upload/10159/10159fig1.jpg" /> 
Figure 1. Geochemically important functional groups. From Killops and Killops1.

Aufreinigung eines Gesamtlipidextrakts mit Säulenchromatographie

Removal of Branched and Cyclic Compounds by Urea Adduction for Uk'37 Paleothermometry

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
As mentioned in previous videos, the product of an organic solvent extraction, a total lipid extract (TLE), is often a complex mixture of hundreds, if not thousands, of different compounds. The researcher is often only interested in a handful of compounds. In the case of our two organic paleothermometers (Uk&#39;37 and MBT/CBT), the interest is in only 6 compounds (2 alkenones and 4 isoprenoidal glycerol dialkyl glycerol tetraethers). As discussed in the previous two videos in this series, purification techniques may be applied in order to pare down the number of compounds in an analyzed sample. These techniques may chemically alter the unwanted components (saponification), take advantage of the different compound chemistries (column chromatography), or use the different shapes and sizes of the molecules to include or exclude certain components from the analysis (urea adduction). The atomic structure of different chemicals leads some organic compounds to form long, narrow, straight chains (n-alkanes and alkenones), other organic compounds to form complex cyclic structures, others to form highly-branched structures, and yet others which form both cyclic and branched structures (GDGTs) (Figure 1). The different shapes and sizes of the compounds in a sample can be used to separate them from one another, in much the same way as a coin sorter separates coins of different denominations (sizes).
<img alt="Figure 1" src="/files/ftp_upload/10160/10160fig1.jpg" /> 
Figure 1. Comparison of different chemical structures. Decane, a straight-chained alkane (A; from from http://www.bpc.edu/mathscience/chemistry), cyclohexane, a cyclic alkane (B; from http://www.bpc.edu/mathscience/chemistry), a steroid, a poly-cyclic hydrocarbon (C; from www.wikiwand.com), and 2,2-dimethylbutane, a branched alkane (D; www.wikimedia.com). <a href="https://www.jove.com/files/ftp_upload/10160/10160fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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