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Carbon-13 nuclear magnetic resonance
Carbon-13 nuclear magnetic resonance
Carbon-13 nuclear magnetic resonance
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Carbon-13 (C13) nuclear magnetic resonance (most commonly known as carbon-13 NMR spectroscopy or 13C NMR spectroscopy or sometimes simply referred to as carbon NMR) is the application of nuclear magnetic resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR (1
H NMR) and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. 13C NMR detects only the 13
C isotope. The main carbon isotope, 12
C does not produce an NMR signal. Although about 1 million times less sensitive than 1H NMR spectroscopy, 13C NMR spectroscopy is widely used for characterizing organic and organometallic compounds, primarily because 1H-decoupled 13C-NMR spectra are simpler, have a greater sensitivity to differences in the chemical structure, and thus are better suited for identifying molecules in complex mixtures.[1] At the same time, such spectra lack quantitative information about the atomic ratios of different types of carbon nuclei, because the nuclear Overhauser effect used in 1H-decoupled 13C-NMR spectroscopy enhances the signals from carbon atoms with a larger number of hydrogen atoms attached to them more than from carbon atoms with a smaller number of H's, and because full relaxation of 13C nuclei is usually not attained (for the sake of reducing the experiment time), and the nuclei with shorter relaxation times produce more intense signals.

The major isotope of carbon, the 12C isotope, has a spin quantum number of zero, so is not magnetically active and therefore not detectable by NMR. 13C, with a spin quantum number of 1/2, is less abundant (1.1%), whereas other popular nuclei are 100% abundant, e.g. 1H, 19F, 31P.

Receptivity

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13C NMR spectroscopy is much less sensitive (ca. by 4 orders of magnitude) to carbon than 1H NMR spectroscopy is to hydrogen, because of the lower abundance (1.1%) of 13C compared to 1H (>99%), and because of a lower(0.702 vs. 2.8) nuclear magnetic moment. Stated equivalently, the gyromagnetic ratio (6.728284 107 rad T−1 s−1) is only 1/4th that of 1H.[2]

On the other hand, the sensitivity of 13C NMR spectroscopy benefits to some extent from nuclear Overhauser effect, which enhances signal intensity for non-quaternary 13C atoms.

Chemical shifts

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The disadvantages in "receptivity" are compensated by the high sensitivity of 13C NMR signals to the chemical environment of the nucleus, i.e. the chemical shift "dispersion" is great, covering nearly 250 ppm. This dispersion reflects the fact that non-1H nuclei are strongly influenced by excited states ("paramagnetic" contribution to shielding tensor. This paramagnetic contribution is unrelated to paramagnetism).[3] For example, most 1H NMR signals for most organic compounds are within 15 ppm.

The chemical shift reference standard for 13C is the carbons in tetramethylsilane (TMS), [4] whose chemical shift is set as 0.0 ppm at every temperature.

Typical chemical shifts in 13C-NMR

Coupling constants

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Homonuclear 13C-13C coupling is normally only observed in samples that are enriched with 13C. The range for one-bond 1J(13C,13C) is 50–130 Hz. Two-bond 2J(13C,13C) are near 10 Hz.

The trends in J(1H,13C) and J(13C,13C) are similar, except that J(1H,13C are smaller owing to the modest value of the 13C nuclear magnetic moment. Values for 1J(1H,13C) range from 125 to 250 Hz. Values for 2J(1H,13C) are near 5 Hz and often are negative.

Implementation

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Sensitivity

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As a consequence of low receptivity, 13C NMR spectroscopy suffers from complications not encountered in proton NMR spectroscopy. Many measures can be implemented to compensate for the low receptivity of this nucleus. For example, high field magnets with wider internal bores are capable of accepting larger sample tubes (typically 10 mm in diameter for 13C NMR versus 5 mm for 1H NMR). Relaxation reagents allow more rapid pulsing.[5] A typical relaxation agent is chromium(III) acetylacetonate. For a typical sample, recording a 13C NMR spectrum may require several hours, compared to 15–30 minutes for 1H NMR. The nuclear dipole is weaker, the difference in energy between alpha and beta states is one-quarter that of proton NMR, and the Boltzmann population difference is correspondingly less.[6] One final measure to compensate for low receptivity is isotopic enrichment.

Some NMR probes, called cryoprobes, offer 20x signal enhancement relative to ordinary NMR probes. In cryoprobes, the RF generating and receiving electronics are maintained at ~ 25K using helium gas, which greatly enhances their sensitivity.[7] The trade-off is that cryoprobes are costly.

Coupling modes

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Another potential complication results from the presence of large one bond J-coupling constants between carbon and hydrogen (typically from 100 to 250 Hz). While potentially informative, these couplings can complicate the spectra and reduce sensitivity. For these reasons, 13C-NMR spectra are usually recorded with proton NMR decoupling. Couplings between carbons can be ignored due to the low natural abundance of 13C. Hence in contrast to typical proton NMR spectra, which show multiplets for each proton position, carbon NMR spectra show a single peak for each chemically non-equivalent carbon atom.[8]

In further contrast to 1H NMR, the intensities of the signals are often not proportional to the number of equivalent 13C atoms. Instead, signal intensity is strongly influenced by (and proportional to) the number of surrounding spins (typically 1H). Integrations are more quantitative if the delay times are long, i.e. if the delay times greatly exceed relaxation times.

The most common modes of recording 13C spectra are proton-noise decoupling (also known as noise-, proton-, or broadband- decoupling), off-resonance decoupling, and gated decoupling. These modes are meant to address the large J values for 13C - H (110–320 Hz), 13C - C - H (5–60 Hz), and 13C - C - C - H (5–25 Hz) which otherwise make completely proton coupled 13C spectra difficult to interpret.[9]

With proton-noise decoupling, in which most spectra are run, a noise decoupler strongly irradiates the sample with a broad (approximately 1000 Hz) range of radio frequencies covering the range (such as 100 MHz for a 23,486 gauss field) at which protons change their nuclear spin. The rapid changes in proton spin create an effective heteronuclear decoupling, increasing carbon signal strength on account of the nuclear Overhauser effect (NOE) and simplifying the spectrum so that each non-equivalent carbon produces a singlet peak.

Both the atoms, carbon and hydrogen exhibit spins and are NMR active. The nuclear Overhauser Effect is in general, showing up when one of two different types of atoms is irradiated while the NMR spectrum of the other type is determined. If the absorption intensities of the observed (i.e., non-irradiated) atom change, enhancement occurs. The effect can be either positive or negative, depending on which atom types are involved.[10]

The relative intensities are unreliable because some carbons have a larger spin-lattice relaxation time and others have weaker NOE enhancement.[9]

In gated decoupling, the noise decoupler is gated on early in the free induction delay but gated off for the pulse delay. This largely prevents NOE enhancement, allowing the strength of individual 13C peaks to be meaningfully compared by integration, at a cost of half to two-thirds of the overall sensitivity.[9]

With off-resonance decoupling, the noise decoupler irradiates the sample at 1000–2000 Hz upfield or 2000–3000 Hz downfield of the proton resonance frequency. This retains couplings between protons immediately adjacent to 13C atoms but most often removes the others, allowing narrow multiplets to be visualized with one extra peak per bound proton (unless bound methylene protons are non-equivalent, in which case a pair of doublets may be observed).[9]

Distortionless enhancement by polarization transfer spectra

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Various DEPT spectra of propyl benzoate
From top to bottom: 135°, 90° and 45°

Distortionless enhancement by polarization transfer (DEPT)[11] is an NMR method used for determining the presence of primary, secondary and tertiary carbon atoms. The DEPT experiment differentiates between CH, CH2 and CH3 groups by variation of the selection angle parameter (the tip angle of the final 1H pulse): 135° angle gives all CH and CH3 in a phase opposite to CH2; 90° angle gives only CH groups, the others being suppressed; 45° angle gives all carbons with attached protons (regardless of number) in phase.

Signals from quaternary carbons and other carbons with no attached protons are always absent (due to the lack of attached protons).

The polarization transfer from 1H to 13C has the secondary advantage of increasing the sensitivity over the normal 13C spectrum (which has a modest enhancement from the nuclear overhauser effect (NOE) due to the 1H decoupling).

Attached proton test spectra

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Another useful way of determining how many protons a carbon in a molecule is bonded to is to use an attached proton test (APT), which distinguishes between carbon atoms with even or odd number of attached hydrogens. A proper spin-echo sequence is able to distinguish between S, I2S and I1S, I3S spin systems: the first will appear as positive peaks in the spectrum, while the latter as negative peaks (pointing downwards), while retaining relative simplicity in the spectrum since it is still broadband proton decoupled.

Even though this technique does not distinguish fully between CHn groups, it is so easy and reliable that it is frequently employed as a first attempt to assign peaks in the spectrum and elucidate the structure.[12] Additionally, signals from quaternary carbons and other carbons with no attached protons are still detectable, so in many cases an additional conventional 13C spectrum is not required, which is an advantage over DEPT. It is, however, sometimes possible that a CH and CH2 signal have coincidentally equivalent chemical shifts resulting in annulment in the APT spectrum due to the opposite phases. For this reason the conventional 13C{1H} spectrum or HSQC are occasionally also acquired.

See also

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References

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[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Carbon-13 nuclear magnetic resonance

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Carbon-13 nuclear magnetic resonance , commonly abbreviated as 13C NMR, is a spectroscopic method that probes the nuclear spin transitions of the in molecules to reveal information about their carbon framework and chemical environments. The nucleus, with a of 1/2, is the only stable carbon suitable for NMR detection, as the predominant (98.93% natural abundance) lacks nuclear spin, and is radioactive. With a low natural abundance of approximately 1.07%, 13C NMR signals are inherently weak, necessitating techniques such as prolonged acquisition times, high magnetic fields, or proton decoupling to achieve detectable spectra. In 13C NMR, the chemical shift of each carbon resonance—typically ranging from 0 to 220 ppm relative to (TMS) as the standard—depends on factors like hybridization, electronegative substituents, and electronic effects, providing a direct map of distinct carbon types within a . For instance, sp³-hybridized carbons appear between 0 and 50 ppm, aromatic carbons between 110 and 150 ppm, and carbonyl carbons between 160 and 210 ppm, offering greater dispersion than proton NMR for . Proton decoupling, achieved by irradiating the sample with a radiofrequency pulse, removes carbon-hydrogen coupling (J_CH ≈ 120-200 Hz), simplifying the spectrum to singlets and enhancing sensitivity through the (NOE), which can boost signal intensity by up to threefold. Developed in the mid-20th century, with pioneering high-resolution spectra reported in the 1950s and 1960s by researchers like , 13C NMR has become indispensable in for elucidating molecular structures, confirming compound identity, and assessing purity. In biochemistry and , it enables non-destructive analysis of metabolic pathways, polymer compositions, and biomolecular dynamics, often complemented by multidimensional techniques like HSQC or INEPT for correlating carbon signals with attached protons. Despite challenges like lower sensitivity compared to 1H NMR, advancements in instrumentation and pulse sequences continue to expand its applications in vivo and solid-state studies.

Fundamentals

Nuclear Properties

Carbon-13 (13C^{13}\mathrm{C}) is a stable isotope of carbon with a mass number of 13 and a natural abundance of 1.07%. It has a nuclear spin quantum number I=12I = \frac{1}{2}, which enables it to interact with magnetic fields in nuclear magnetic resonance (NMR) spectroscopy. In contrast, the predominant isotope 12C^{12}\mathrm{C}, with I=0I = 0, is NMR-inactive and constitutes over 98.9% of naturally occurring carbon, meaning 13C^{13}\mathrm{C} NMR primarily probes the carbon framework of organic molecules through this minor isotopic component. The gyromagnetic ratio γ\gamma of 13C^{13}\mathrm{C} is 10.705 MHz/T, about one-fourth that of the proton (1H^{1}\mathrm{H}, γ=42.577\gamma = 42.577 MHz/T), influencing the strength of its magnetic moment. The resonance frequency, or Larmor frequency, for 13C^{13}\mathrm{C} nuclei is determined by the equation ν=γB0\nu = \gamma B_0, where B0B_0 is the strength of the external magnetic field. This results in lower resonance frequencies for 13C^{13}\mathrm{C} compared to 1H^{1}\mathrm{H} at the same field strength; for instance, at B0=9.4B_0 = 9.4 T (corresponding to a 400 MHz proton spectrometer), the 13C^{13}\mathrm{C} frequency is approximately 100 MHz. These nuclear parameters define the operational regime for 13C^{13}\mathrm{C} NMR experiments, requiring radiofrequency pulses tuned to this lower frequency range. In 13C^{13}\mathrm{C} NMR, the nuclear spins experience Zeeman splitting in the B0B_0, producing two energy levels for I=12I = \frac{1}{2}. Radiofrequency pulses at the Larmor induce transitions between these levels, generating a detectable . Upon perturbation, the system relaxes back to equilibrium through longitudinal relaxation (characterized by time constant T1T_1) and transverse relaxation (characterized by time constant T2T_2); for 13C^{13}\mathrm{C}, these times are generally longer than for protons due to the smaller and reduced dipolar interactions in typical organic environments.

Receptivity

In , the receptivity of a nucleus quantifies its detectability and is proportional to the natural isotopic abundance multiplied by the intrinsic sensitivity factor γ³ I(I + 1), where γ is the and I is the . For ¹³C (I = ½), this yields a relative receptivity of 1.70 × 10⁻⁴ compared to ¹H at natural abundance. The low receptivity of ¹³C stems primarily from its low natural abundance of 1.07%, which reduces the number of observable nuclei in a typical sample by over 99-fold relative to ¹H. Additionally, the of ¹³C (6.728 × 10⁷ rad s⁻¹ T⁻¹) is only about 25% that of ¹H (26.752 × 10⁷ rad s⁻¹ T⁻¹), resulting in a smaller equilibrium magnetization M₀ ∝ γ² and overall signal strength ∝ γ³. Furthermore, ¹³C nuclei exhibit longer longitudinal relaxation times T₁, typically ranging from 1 to 100 seconds in organic molecules, compared to ~1 second for ¹H, which limits the repetition rate of pulse sequences and further diminishes signal averaging efficiency. These factors necessitate practical adjustments in ¹³C NMR experiments, such as extended acquisition times, higher sample concentrations, or reliance on signal enhancement techniques to achieve adequate signal-to-noise ratios. For instance, routine ¹³C spectra often require to 4096 scans (taking ~1–4 hours), in contrast to 8–32 scans (minutes) for ¹H spectra under similar conditions. Historically, early ¹³C NMR studies in the and were severely constrained by this low receptivity when using continuous-wave detection methods, often requiring impractical acquisition times for even basic spectra. The development of NMR in the late and its widespread adoption in the dramatically enhanced sensitivity through efficient signal averaging, rendering ¹³C NMR a feasible routine tool in chemical analysis.

Spectral Features

Chemical Shifts

In carbon-13 nuclear magnetic resonance (¹³C NMR) , chemical shifts are measured relative to (TMS), which is assigned a value of 0 ppm, providing a universal reference standard for organic compounds. The typical range for ¹³C chemical shifts in organic molecules spans approximately 0 to 220 ppm downfield from TMS, allowing for clear distinction of different carbon environments. For instance, methyl carbons in alkanes resonate between 10 and 25 ppm, while carbonyl carbons in ketones and carboxylic acids appear much further downfield at 160 to 220 ppm. Several key factors influence ¹³C chemical shifts, primarily through alterations in the local magnetic shielding around the nucleus. Electronegative substituents attached to or near the carbon atom induce deshielding via inductive effects, shifting resonances downfield; for example, an α-effect from an electronegative group like oxygen or a halogen can displace the signal by 10 to 20 ppm per substituent. Hybridization of the carbon atom plays a dominant role, with sp³-hybridized carbons (e.g., in alkanes) typically appearing at 0 to 70 ppm, sp²-hybridized carbons (e.g., in alkenes and aromatics) at 100 to 150 ppm, and sp-hybridized carbons (e.g., in alkynes) at 70 to 110 ppm. In unsaturated systems, magnetic anisotropy from π-electron clouds or nearby multiple bonds further modulates shifts, often causing additional deshielding in the plane of the double bond. Empirical rules facilitate the prediction of ¹³C chemical shifts, particularly for aliphatic hydrocarbons. The Grant-Paul rules, developed for alkanes, use additivity parameters based on positions relative to the observed carbon. Base shifts are approximately 7 ppm for terminal methyl (CH₃-) groups and 16 ppm for methylene (-CH₂-) groups, with adjustments from α-effects (+9 ppm for adjacent s), β-effects (+9 ppm for substituents two bonds away), and γ-effects (-3 ppm for substituents three bonds away). These parameters account for long-range influences and branching, enabling shift estimates within a few ppm for simple alkanes. Solvent and concentration effects can perturb ¹³C chemical shifts by 1 to 5 ppm or more, depending on polarity and hydrogen-bonding interactions. In nonpolar solvents like CDCl₃, aromatic carbons typically resonate at lower ppm values compared to polar solvents such as D₂O, where β- and γ-carbons in shift downfield by up to 1.5 ppm relative to CDCl₃ due to enhanced deshielding from . Higher concentrations may amplify these variations through intermolecular associations, though effects are generally smaller for ¹³C than for ¹H NMR. Modern computational approaches, such as (DFT) with the gauge-including (GIAO) method, enable accurate prediction of ¹³C chemical shifts for structural assignment, often within ±5 ppm of experimental values. These calculations incorporate solvent models and electron correlation to reproduce , hybridization, and influences, supporting the interpretation of complex spectra in and analysis.
Carbon TypeTypical Shift Range (ppm)Example
Aliphatic sp³ (methyl)10–25CH₃ in
Aliphatic sp³ (methylene)20–40-CH₂- in
Olefinic sp²100–150=CH- in ethene
Aromatic sp²110–150 ring carbons
Alkyne sp70–110-C≡CH in
Carbonyl sp²160–220C=O in acetone

Coupling Constants

In carbon-13 nuclear magnetic resonance (¹³C NMR) , spin-spin coupling constants (J values) arise from the interaction between nuclear through electrons, providing key insights into molecular connectivity and . These couplings are particularly important for identifying carbon-hydrogen attachments and long-range relationships, influencing the multiplicity and appearance of peaks. The dominant couplings in ¹³C NMR are the one-bond heteronuclear couplings between ¹³C and directly attached ¹H, denoted as ¹J_CH, which typically range from 120 to 200 Hz. This value correlates with carbon hybridization: approximately 125 Hz for sp³-hybridized CH groups in alkanes, 145-170 Hz for sp²-hybridized CH in alkenes, and up to 250 Hz for sp-hybridized CH in alkynes. These ¹J_CH couplings determine the multiplicity in proton-coupled ¹³C spectra, where a methine (CH) carbon appears as a doublet split by ~125-170 Hz, a methylene (CH₂) as a triplet, and a methyl (CH₃) as a , enabling direct assessment of the number of attached protons. Longer-range ¹³C-¹H couplings are smaller and more variable. Two-bond couplings (²J_CH) generally fall between 0 and 10 Hz, while three-bond vicinal couplings (³J_CH) range from 0 to 12 Hz and exhibit a Karplus-type dependence on the θ, approximated by the relation
3J\ceCH≈9cos⁡2θ−cos⁡θ+0.3^3J_\ce{CH} \approx 9 \cos^2 \theta - \cos \theta + 0.3 Hz, which is useful for probing torsional conformations in flexible molecules. Four-bond and longer-range ¹³C-¹H couplings are typically under 5 Hz and often contribute to complex multiplet . Other heteronuclear couplings involving ¹³C include those with adjacent ¹³C atoms (¹J_CC), which are small (0-50 Hz, often 30-50 Hz for sp³ carbons) and rarely resolved in natural-abundance spectra due to the 1.1% isotopic abundance of ¹³C, manifesting as weak peaks flanking the main signal. In molecules containing or , ¹J_CN couplings (typically 10-20 Hz, e.g., 20 Hz in ) or ¹J_CF couplings (often 200-300 Hz, e.g., 230-250 Hz in fluorocarbons) can significantly split ¹³C resonances, providing structural information in specific contexts like amides or fluoroorganics. Precise measurement of these J values is achieved through techniques such as selective ¹H decoupling to isolate specific couplings or two-dimensional experiments like HSQC for ¹J_CH (where the splitting in the ¹H dimension directly reflects the coupling) and HMBC for long-range ³J_CH. The magnitude of ¹J_CH, for instance, correlates with hybridization and dihedral angles, enhancing its utility in structural elucidation. In standard proton-decoupled ¹³C NMR, broadband decoupling eliminates ¹³C-¹H couplings, yielding singlets for all carbon signals and simplifying interpretation, though at the cost of multiplicity data essential for connectivity determination.

Experimental Implementation

Sensitivity Considerations

One key strategy to enhance the inherently low sensitivity of ^{13}C NMR involves exploiting the (NOE) through broadband proton irradiation during signal acquisition. This heteronuclear NOE transfers polarization from abundant protons to ^{13}C nuclei, yielding an enhancement factor η of approximately 1 + \frac{\gamma_H}{2 \gamma_C} under extreme narrowing conditions, resulting in a typical signal boost of about 2-fold, though maximum values up to 3-fold are achievable for proton-bearing carbons. However, the actual enhancement ranges from 0 to 3 depending on molecular correlation times and motion, with carbons showing minimal or no gain. Hardware optimizations further address sensitivity limitations. Cryogenically cooled probes reduce thermal noise in the radiofrequency coils, providing a signal-to-noise (S/N) improvement of up to 4-fold for ^{13}C detection compared to room-temperature probes, particularly beneficial for organic solvents. Higher strengths, such as 600 MHz versus 300 MHz instruments, enhance S/N by a factor scaling with the^{3/2} power of the field ratio (approximately 2.8-fold for doubled field), enabling faster acquisitions or better resolution for dilute samples. also plays a critical role, with optimal concentrations of 0.5-1 M in standard 5 mm tubes maximizing fill height and S/N while avoiding viscosity-induced line broadening. Acquisition parameters must balance sensitivity and experimental duration. A spectral width of approximately 200 ppm accommodates the broad chemical shift range of carbon environments, from alkyl to carbonyl regions. Relaxation delays are typically set to 1-5 times the longest ^{13}C spin-lattice relaxation time (T_1), often 20-60 seconds for quantitative measurements to ensure full recovery, with the number of scans adjusted as a against total time—higher scans improve S/N linearly with the but extend acquisition. For quantitative ^{13}C NMR, where accurate peak integrals reflect carbon abundances, inverse gated decoupling is essential. This technique applies proton decoupling only during acquisition, minimizing NOE variability across sites and preventing differential enhancements that distort integrals, thereby enabling reliable quantification even with varying . A significant recent advancement is dissolution dynamic nuclear polarization (DNP), which hyperpolarizes ^{13}C nuclei at low temperatures before rapid dissolution into solution, achieving enhancements exceeding 1000-fold for natural-abundance samples. Developed in the early and refined since, this method has enabled high-sensitivity studies of low-concentration metabolites in biofluids, dramatically reducing acquisition times for otherwise impractical experiments.

Decoupling Modes

In carbon-13 nuclear magnetic resonance (¹³C NMR) , decoupling modes are employed to remove heteronuclear scalar couplings, primarily between ¹³C and ¹H nuclei, thereby simplifying complex multiplet patterns and enhancing . These techniques involve irradiating the sample with radiofrequency (RF) pulses at the ¹H to average the spin states of attached protons, collapsing ¹³C signals into singlets or reduced multiplets. decoupling is the most common approach, utilizing composite sequences such as WALTZ-16 or GARP to apply ¹H irradiation across the typical range of 0-10 ppm, effectively eliminating one-bond J_CH couplings (typically 120-200 Hz) and producing singlets for all carbon types while increasing signal intensity through the (NOE). Off-resonance decoupling, a predating advanced pulse sequences, involves partial ¹H offset from the proton , resulting in residual splittings with reduced constants. This produces characteristic patterns—such as quartets for CH₃, triplets for CH₂, doublets for CH, and singlets for C—facilitating the identification of carbon hybridization without full decoupling. Although largely superseded by modern techniques for routine analysis, it remains useful in specific cases for determining the number of directly attached protons. Gated decoupling combines elements of coupled and decoupled spectra by applying continuous ¹H irradiation during the relaxation delay but turning it off during signal acquisition, preserving J_CH couplings for structural insight while allowing NOE enhancement to build up during the delay for improved sensitivity. This mode is particularly valuable for quantitative applications where coupling information is needed, such as measuring relative peak intensities in coupled spectra without NOE bias distortion, though longer acquisition times are required compared to fully decoupled modes. Selective decoupling targets irradiation to specific proton resonances, enabling the measurement of individual J_CH couplings or assignment of nearby carbons without affecting the entire spectrum. In natural product studies, for example, selective irradiation of a methine proton can collapse the corresponding ¹³C doublet, confirming connectivity and aiding complex structure elucidation. This technique requires precise frequency selection and is often implemented with shaped pulses for efficiency. Despite their utility, decoupling modes introduce artifacts and limitations, including sample heating from high-power RF , which can cause temperature gradients and line broadening, particularly in aqueous or conductive samples. Incomplete decoupling may occur for large J_CH values if the irradiation bandwidth is insufficient, leading to residual splittings, while early continuous-wave methods suffered from uneven coverage across the proton range—addressed by composite pulses like WALTZ-16. Additionally, decoupling sidebands or artifacts can appear near intense signals, necessitating careful power calibration and sequence optimization.

Advanced Pulse Sequences

Distortionless Enhancement by Polarization Transfer (DEPT)

Distortionless Enhancement by Polarization Transfer (DEPT) is a heteronuclear pulse sequence that boosts the sensitivity of 13^{13}C NMR spectroscopy by transferring magnetization from 1^{1}H to 13^{13}C nuclei through the Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) mechanism, while maintaining phase coherence to produce distortionless multiplets. Introduced in 1982, DEPT exploits the larger gyromagnetic ratio and higher natural abundance of protons to enhance 13^{13}C signals, typically achieving a 2-4 fold increase in sensitivity compared to conventional proton-decoupled 13^{13}C NMR, owing to the polarization transfer efficiency and the shorter 1^{1}H longitudinal relaxation times (T1T_1) that permit shorter repetition delays. This transfer relies on the one-bond 1J\ceCH^{1}J_{\ce{CH}} coupling constants, typically 120-200 Hz, to create antiphase magnetization on 13^{13}C during the evolution periods. The core DEPT pulse sequence modifies the standard INEPT framework by incorporating a variable flip angle θ\theta on the final 1^{1}H pulse, which controls the phase and visibility of signals based on the number of attached protons (nn). It begins with a 90∘^\circ pulse on 1^{1}H to create transverse magnetization, followed by a delay τ=1/(21J\ceCH)\tau = 1/(2^{1}J_{\ce{CH}}) for evolution into antiphase magnetization, a simultaneous 180∘^\circ pulse pair on 1^{1}H and 13^{13}C to refocus chemical shifts, another τ\tau delay to generate zero-quantum and double-quantum coherences, a 90∘^\circ pulse on 13^{13}C to transfer polarization, the variable θ\theta pulse on 1^{1}H (e.g., θ=45∘\theta = 45^\circ, 90∘^\circ, or 135∘^\circ), and finally acquisition of the 13^{13}C signal under broadband 1^{1}H decoupling. For θ=45∘\theta = 45^\circ (DEPT-45), all protonated carbons (CH, CH2_2, CH3_3) yield positive-phase signals with intensities scaled by sin⁡(θ)n\sin(\theta)^n. In DEPT-90 (θ=90∘\theta = 90^\circ), only methine (CH) carbons appear as positive singlets, as the transfer efficiency for n>1n > 1 drops to zero. DEPT-135 (θ=135∘\theta = 135^\circ) provides spectral editing where CH and CH3_3 signals are positive, CH2_2 signals are negative (inverted phase), and quaternary (C) carbons remain invisible, enabling clear distinction of carbon environments by proton attachment. Relaxation considerations are critical, as the short 1^{1}H T1T_1 (often 1-5 s) supports rapid pulsing, but 13^{13}C T1T_1 and 1J\ceCH^{1}J_{\ce{CH}} variations can affect quantitative accuracy unless optimized. DEPT's primary advantages lie in its sensitivity enhancement and multiplicity editing, which facilitate 13^{13}C signal assignments in low-concentration samples without the peak overlap issues of 1^{1}H NMR. This has proven invaluable for analyzing complex structures, such as determining carbon types in synthetic polymers like copolymers of styrene and butadiene, where DEPT distinguishes CH2_2 chain segments from aromatic CH. In natural product chemistry, DEPT aids in elucidating skeletal frameworks, as seen in the assignment of protonated carbons in alkaloids and terpenoids, streamlining dereplication and quantification efforts. By suppressing quaternary signals in standard variants, DEPT focuses on proton-bearing carbons, though this can be a limitation for fully substituted centers. Several variants address these gaps while building on the DEPT framework. DEPTQ (DEPT including quaternary carbons) adds a 135∘^\circ 13^{13}C pulse after the standard sequence to refocus and display carbons as positive singlets, enabling complete 13^{13}C editing in a single experiment with minimal sensitivity loss. Another extension, (Polarization Enhancement Nurtured During Attached Nucleus Testing), modifies the transfer to enhance quaternary signals and probe stereochemical differences through selective polarization pathways in chiral environments.

Attached Proton Test (APT)

The Attached Proton Test (APT) is a one-dimensional ^{13}C (NMR) pulse that distinguishes protonated carbons (CH and CH_3) from non-protonated ones (CH_2 and C) through phase editing, providing a of carbon multiplicities in organic molecules. Developed in the early as a simple alternative to emerging spectral editing techniques, APT relies on a spin-echo framework to exploit the one-bond carbon-proton scalar (^{1}J_{CH}), typically 120-140 Hz in aliphatic and aromatic systems, without requiring polarization transfer. This approach causes a characteristic phase alternation in the spectrum: signals from CH and CH_3 groups appear positive (upright), while those from CH_2 and carbons appear negative (inverted), based on the evolution of antiphase over the coupling period, modulated by the factor \sin(\gamma J \tau / 2\pi) where \tau is the evolution delay and \gamma is the difference. The implementation of APT is straightforward and spin-echo based, consisting of an initial low-flip-angle excitation pulse on ^{13}C (often \theta \approx 45^\circ for optimal sensitivity under conditions), followed by a delay \tau = 1/(2 ^{1}J_{CH}) \approx 3.6-4.0 ms for an average ^{1}J_{CH} of 125 Hz, then simultaneous 180^\circ refocusing pulses on both ^{13}C and ^1H nuclei, another delay \tau, and finally signal acquisition under broadband ^1H decoupling to collapse multiplets. In some variants, phase cycling with \theta = 180^\circ / 2^n (where n relates to acquisition steps) enhances artifact suppression, but the core sequence ensures chemical shift refocusing while allowing J-coupling evolution to dictate signal polarity. The resulting spectrum inverts quaternary and CH_2 signals relative to a standard broadband-decoupled ^{13}C trace, facilitating rapid multiplicity assignment without additional experiments. APT offers several advantages for routine ^{13}C NMR analysis, including its simplicity—no variable flip angles or multi-scan variants are needed, unlike polarization transfer methods—while detecting all carbon types, including low-sensitivity centers that often evade other editing sequences. Its sensitivity matches that of direct ^{13}C observation with nuclear Overhauser enhancement (NOE) from protons, making it ideal for quick structural surveys in small samples, as demonstrated in spectra of compounds like where the polarity flip clearly separates CH/CH_3 (positive) from CH_2/C (negative) peaks. Despite these benefits, APT has limitations stemming from its reliance on uniform ^{1}J_{CH} values; deviations, such as lower couplings in certain CH_2 groups (e.g., \approx 100-110 Hz in some aromatics or strained systems), can cause partial phase inversion or signal , reducing accuracy. Artifacts from long-range couplings (^{2}J_{CH} or ^{3}J_{CH} > 2-3 Hz) may also distort phases, particularly in complex molecules, though these are often minor in protonated carbons. Example spectra, such as those from , illustrate the clean polarity flip under ideal conditions but highlight distortions in heterogeneous J environments. The technique was pioneered by Patt and Shoolery in 1982 as an accessible tool for ^{13}C multiplicity , quickly adopted for its efficiency in early NMR .

References

  1. https://www1.udel.edu/chem/kohclasses/docs/13CNMRfor322.pdf
  2. https://physics.nist.gov/cgi-bin/Compositions/stand_alone.pl?ele=C
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