Pulse Sequences

Distortionless Enhancement by Polarisation Transfer (DEPT)

The DEPT sequence uses a polarisation transfer from protons to an X-nucleus (usually carbon-13 or Silicon-29), i.e. exciting the protons and observing the NMR signal from the X-nuclei; thereby increasing the signal strength in comparison with the standard one-dimensional NMR spectra. Additionally the sequence can be using to distinguish signals depending on the number of protons coupled to the nucleus, by varying the duration of the RF pulses (usually described as the flip angle), and the length of delays between the pulses in the sequence (which correlate with the proton–X-nuclei J-couplings). 

¹³C{¹H} DEPT commonly uses flip angles of 45°, 90° & 135°, to distinguish between CH₃, CH₂, CH and quaternary-carbons. In a DEPT-45 spectrum CH₃, CH₂ and CH signals are observed, all with the same phase; while in a DEPT-90 spectrum only CH signals are observed; finally in a DEPT-135 spectrum CH₃, CH₂ and CH signals are observed, though CH₂ signals have opposite phase to CH₃ and CH signals. Together with a standard ¹³C{¹H} spectrum, this permits the different CHₓ signals to be distinguished.

Meanwhile for ²⁹Si{¹H} DEPT appropriate choices of flip angles, and proton-silicon J-couplings (¹JSiH ≈ 200 Hz, ²JSiH ≈ 10 Hz), allow for selective observation of differing silicon environments within molecules. For example a DEPT-90 spectrum of 1,1,3,3,5,5-hexamethyltrisiloxane, with delays correlating with a J-coupling of 200 Hz, only shows a single peak corresponding to the two SiH silicon atoms; while a DEPT-24 spectrum, with delays correlating with a J-coupling of 10 Hz, will only show peaks corresponding to Si(CH₃)₂ environments, which are both of the chemically unique silicon environments in 1,1,3,3,5,5-hexamethyltrisiloxane.

²⁹Si{¹H} DEPT-24 & DEPT-90 spectra of 1,1,3,3,5,5-hexamethyltrisiloxane in benzene-d₆

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¹³C{¹H} DEPT, Quinine in DMSO-d₆

Pulsed field Gradient Spin-Echo (PGSE)

Pulsed field Gradient Spin-Echo (PGSE) experiments are a convenient method for measuring diffusion constants by NMR spectroscopy. The sequence incorporates pulsed field gradients, and as the power of these gradients is increased (to at least 0.25 T.m⁻¹ on the X-Pulse), the NMR signals are attenuated. 

The diffusion constant is calculated from this effect using the Stejskal-Tanner equation (E. O. Stejskal & J. E. Tanner, J. Chem. Phys., 1965, 42, 288-292), which also takes into account the properties of the nuclei of interest, and the duration of / delay between the gradient pulses.

The PGSE sequence is available on the X-Pulse for all supported nuclei; with optional J-compensation in the proton/fluorine version, which can overcome difficulties in phasing the resulting spectra.

Series of ¹⁹F PGSE spectra of Li[PF₆] in alkyl carbonates

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⁷Li PGSE, Li[PF₆] in PC

Inversion Recovery (InvRec)

The Inversion Recovery (InvRec) pulse sequence in used for the measurement of spin-lattice relaxation time (T₁). The InvRec sequence comprises two RF pulses separated by a time, τ; by collecting a series of InvRec spectra with increasing values of τ, and phasing the spectra identically, the phase of each signal will invert as τ increases, from this T₁ can be calculated for each signal in the NMR spectrum. 

The InvRec sequence is available on the X-Pulse for all supported nuclei.

Series of ¹H InvRec spectra of Ethyl Crotonate in CDCl₃

Carr-Purcell-Meiboom-Gill (CPMG)

The Carr-Purcell-Meiboom-Gill (CPMG) sequence is one of the few NMR pulse sequence actually named after its creators. Amongst its applications is the measurement of spin-spin relaxation (T₂) times; collecting a series of spectra with increasing delays prior to acquisition, and by measuring the attenuation of each signal as the delay increases, T₂ can be calculated for each signal. The CPMG sequence can also be used to selectively observe signals with long T₂ times within a mixture; for example, unreacted monomers (small molecules with relatively long T₂) in a polymer sample (large molecules with generally short T₂). 

The CPMG sequence is available on the X-Pulse for all supported nuclei.

Series of ¹H CPMG spectra of Ethyl Crotonate in CDCl₃

Correlation Spectroscopy (COSY)

COSY was one of the first two-dimensional NMR experiments developed, and remains amongst the most popular, with a number of variants. The spectrum obtained from a COSY experiment, shows frequencies for a single isotope along both axis (depending on the configuration of the spectrometer, it is also possible to obtain COSY spectra of two different isotopes – if they’re observed on the same channel); peaks observed along the diagonal correspond to the peaks in the standard one-dimensional NMR experiment; while the off-diagonal cross-peaks indicate couplings between pairs of nuclei (in general if a J-coupling is observed in the one-dimensional spectrum – in the form of multiplets – corresponding cross-peaks will be observed in a COSY spectrum). 

COSY spectra have been obtained on the X-Pulse to observe proton-proton, fluorine-fluorine, and proton-fluorine correlations.

¹H-¹H gs-COSY, 3-Dimethylaminoacrolein in CDCl₃ at +20°C

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¹⁹F-¹⁹F gs-COSY, Mixture of 1-Bromo-2,4,5-trifluorobenzene and 5-Bromo-1,2,3-trifluorobenzene in CDCl₃

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¹H-¹⁹F gs-COSY, 1,4-difluoro-2,5-dimethoxybenzene in CDCl₃

Total Correlation Spectroscopy (TOCSY)

While the COSY spectrum shows only peaks arising from direct (usually three-bond) couplings, a TOSCY spectrum shown both direct and indirect couplings. 
For example, consider a hydrocarbon chain including four hydrogen atoms, labelled Hᵃ, Hᵇ, Hᶜ & Hᵈ; where Hᵃ is coupled to Hᵇ which is coupled to Hᶜ which, in turn, is coupled to Hᵈ. In a COSY experiment we would expect to see a cross-peak between hydrogen nuclei in the following pairs of positions, (Hᵃ, Hᵇ), (Hᵇ, Hᶜ) & (Hᶜ, Hᵈ). In contrast, the TOCSY spectrum of this system would show cross-peaks between all pairs of nuclei, i.e. (Hᵃ, Hᵇ), (Hᵇ, Hᶜ), (Hᶜ, Hᵈ), (Hᵃ, Hᶜ), (Hᵃ, Hᵈ) & (Hᵇ, Hᵈ). 

Hence, while the ¹H-¹H COSY of 3-dimethylaminoacrolein, only showed cross-peaks corresponding to three-bond proton-proton correlations; the TOCSY spectrum of the same sample shows cross-peaks corresponding to not just the direct (three-bond) couplings, but also cross-peaks corresponding to the indirect (in this case four-bond) coupling along the hydrocarbon backbone. 

¹H-¹H gs-TOCSY, 3-Dimethylaminoacrolein in CDCl₃ at +20°C 

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¹H-¹H gs-TOCSY, methyl 3-methyl-2-butenoate in CDCl₃

Heteronuclear Multiple Bond Correlation (HMBC)

While the HSQC experiment shows peaks corresponding to correlations through a single bond, the HMBC experiment detects heteronuclear correlations over a longer range of between two and four bonds; this provides information on the connectivity of a molecule and allows for the structural elucidation of an unknown compound. 

As with the HSQC experiment, it’s often quicker to obtain a ¹H-¹³C HMBC two-dimensional spectrum, than it would be to obtain the corresponding standard ¹³C{¹H} one-dimensional spectrum. 

¹H-¹³C gs-HMBC, Ibuprofen in (CD₃)₂SO

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¹H-¹³C gs-HMBC, Gemfibrozil in CDCl₃

Nuclear Overhauser Enhancement Spectroscopy (NOESY)

While both COSY and TOCSY spectra show correlations arising from J-couplings (a through-bond effect); the NOESY experiment uses the nuclear overhauser effect (a through-space effect) to identify signals arising from nuclei which are close in space. The NOESY experiment is therefore the preferred method by which the three-dimensional conformation and stereochemistry of molecules in solution can be identified. 

In the case of methyl 3-methyl-2-butenoate, the ¹H-¹H NOESY spectrum can be used to distinguish the cis and trans alkane bound methyl groups, since only one of them is sufficiently close to the alkane proton for a cross-peak to be observed by NOESY (while in both COSY & TOCSY cross-peaks are observed for both methyl groups with the alkane proton, due to the through-bond J-coupling).

¹H-¹H gs-NOESY, methyl 3-methyl-2-butenoate in CDCl₃

Heteronuclear Single Quantum Coherence with Multiplicity Editing (HSQC-ME)

The HSQC experiment detects correlations between two heteroatoms separated by a single bond, and is often used to correlate proton and carbon-13 signals, aiding in the assignment of the signals in the respective one-dimensional spectra. On the X-Pulse the HSQC sequence is extended as a multiplicity edited experiment; this combines the HSQC two-dimensional experiment, with the information on the nature of the carbon environment which would be obtained from a DEPT-135 spectrum, i.e. signals arising from a CH₂ have opposite phase to those from CH₃ and CH (light-blue and red respectively in the spectra below). 

Since the HSQC experiment, entails both exciting and observing the NMR signal of the high receptivity protons, rather than the low receptivity carbon-13; it’s often quicker to obtain a ¹H-¹³C HSQC two-dimensional spectrum, than it would be to obtain the corresponding standard ¹³C{¹H} one-dimensional spectrum. 

¹H-¹³C gs-HSQC-ME, Ibuprofen in (CD₃)₂SO

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¹H-¹³C gs-HSQC-ME, Gemfibrozil in CDCl₃