Single-shot titrations and reaction monitoring by slice-selective NMR spectroscopy

Abstract

A new method, based on slice-selective NMR spectroscopy of inhomogeneous mixtures, is introduced to perform NMR titrations and reaction monitoring in a single experiment. The method was applied to the titration of a lithium salt with 12-crown-4, and to the reaction of nBuLi with N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDTA).

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NMR spectroscopy plays an increasingly important role in the elucidation of structural and dynamic features of inorganic, organic and biomolecular compounds and their interactions. For example, chemical shift titration is a powerful method to determine the stoichiometry and stability constants of complexes in coordination, supramolecular and medicinal chemistry.^1–3 In such an experiment, the chemical shift of a particular resonance attributed to one component (e.g. the metal, host or target) is monitored in a series of NMR spectra, while the concentration of the other component (e.g. the guest or ligand) is systematically varied. In a different approach, NMR is applied to investigate chemical reactions by monitoring resonances of substrates and products in a series of individual NMR spectra over time. The observation of fast reactions and short-lived intermediates^4,5 has become possible through custom build NMR hardware and techniques such as stopped flow^6,7 and rapid-injection NMR.^8–12

In our research group slice-selective NMR spectroscopy has lately become an important technique to study diffusion of solvents and solutes into polymers.¹³ Using standard solution NMR instrumentation, slice-selection is accomplished by shaped radiofrequency pulses with variable frequency offsets in the presence of a magnetic field gradient along the axis of the NMR tube.^13–17 For highly sensitive nuclei such as ¹H, ⁷Li, ¹⁹F or ³¹P, a series of typically ∼20 conventional NMR spectra of individual horizontal (∼1 mm) slices within the active sample volume (∼2 cm) is obtained in less than 2 min. Here we propose a fast chemical shift titration method based on slice-selective NMR spectroscopy in combination with a concentration gradient of the ligand component rather than incrementing the concentration step-by-step. Alternatively, chemical reactions between two substrates diffusing towards each other may be monitored.

As a case study for the slice-selective titration method we investigated the complexation of a ⁷Li ion with 12-crown-4. For this purpose, 12-crown-4 (50 μL, 3.1 mmol, m.p. 16 °C) was filled into a standard 5 mm NMR tube and cooled to 5 °C. Then a solution of LiClO₄ (24 mg, 2.3 mmol) in acetonitrile-d₃ (0.45 mL) was layered on top of the solid ether. This procedure prevents initial mixing of both components prior to the measurements. Inside the NMR magnet (25 °C, standing tube) the ether melts and slowly diffuses into the LiClO₄ solution resulting in a smooth concentration gradient along the tube axis. Diffusion of LiClO₄ into the ether phase occurs likewise, but with little impact on the ⁷Li concentration due to the 9 : 1 volume ratio. Likewise, the impact of molecular diffusion during the gradient pulse is negligible. Approximately 3, 6 and 9 h after sample preparation slice-selective ¹H and ⁷Li NMR measurements were performed (see ref. 13 and ESI† for details).¹³ For each slice, absolute integrals of both, the ⁷Li resonance of LiClO₄ and the ¹H resonance of 12-crown-4, were measured and converted into concentrations using homogenous reference samples. This way, the ⁷Li chemical shift observed in each slice can be assigned to a specific ether/lithium ratio.

Fig. 1 shows the series of 19 slice-selective ⁷Li NMR spectra recorded after 6 h (for spectra recorded after 3 and 9 h see ESI†). Additional slice-selective ¹H spectra (see also ESI†) confirm that 12-crown-4 diffuses from the bottom to the top and builds up a smooth gradient, while the LiClO₄ concentration is approximately constant. Slice 1 (at the top of the active volume) shows a narrow ⁷Li resonance at −2.3 ppm, typical for Li⁺ coordinated by four acetonitrile molecules. As the ether concentration increases, the ⁷Li resonance is shifted downfield and reaches a maximum (−0.8 ppm) in slice 12, where the 12-crown-4 and LiClO₄ concentrations are approximately equal and thus the [Li(12-crown-4)]⁺ complex dominates.¹⁸ In the presence of an excess of 12-crown-4, the ⁷Li resonance is then shifted again upfield until it reaches −1.2 ppm in slice 19 (7-fold excess, [Li(12-crown-4)₂]⁺). The resulting titration curve (see ESI†) fully agrees with previous reports. Note that the ⁷Li resonances in slices 6 to 11 are increasingly broadened due to (rectangular weighted) summation over the ether concentration gradient within each slice. This broadening could be overcome by further decreasing the slice width, thereby increasing the spatial resolution along the tube axis, but at the cost of reduced sensitivity, which may render weaker signals undetectable.^18,19

Fig. 1 Slice-selective ⁷Li NMR spectra of LiClO₄ in acetonitrile-d₃ in the presence of a concentration gradient of 12-crown-4, 6 h after sample preparation. Generally, slices (1 mm thick, 1 mm distance each) are numbered from the top of the active volume to the bottom, with slice 10 located at the centre. The 12-crown-4/LiClO₄ concentration ratio is indicated on the right and increases from slice 1 to slice 19.

The application of slice-selective NMR spectroscopy to reaction monitoring is demonstrated with the reaction between nBuLi and PMDTA. Due to the high reactivity of organolithium reagents and to prevent rapid convection and/or diffusion out of the active sample volume, nBuLi was first diffused into a polystyrene gel matrix, and all steps of the preparation were carried out strictly under argon atmosphere. For this purpose, a solution of nBuLi in n-hexane (0.4 mL, 1.9 M, 0.74 mmol) was concentrated in vacuo and toluene-d₈ (0.40 mL) was added. This solution was then transferred into a 5 mm NMR tube, and a cylindrical polystyrene stick (10 × 3.8 mm, crosslinked through 0.2 vol% divinylbenzene, for preparation see ref. 20) was immersed in the solution approximately 1 cm above the bottom of the tube. After 7 days (the polystyrene stick had swollen to a length of ∼3 cm) the supernatant nBuLi–toluene solution above the polymer was removed and replaced by a solution of PMDTA (0.12 mL, 0.60 mmol) in toluene-d₈ (0.15 mL). Slice-selective ¹H and ⁷Li NMR measurements started after ∼3 h and went on for three days.

Fig. 2 (bottom left) shows the slice-selective ⁷Li NMR spectra three days after the addition of PMDTA to the polymer imbibed with nBuLi (for spectra recorded after 3 h, 1 and 2 days, see ESI†). Slices 19 and 18 at the bottom of the active volume still show a signal for unreacted nBuLi at 2.3 ppm, in agreement with the absence of ¹H signals of PMDTA in these slices (see also ESI†). On the other hand, in slices 1–12 a narrow resonance at 1.1 ppm is observed, which was assigned to the product of the reaction, lithiated PMDTA (see Fig. 2 top). Satellite peaks in slices 6–12 arise from residual quadrupolar couplings (RQCs) due to partial orientation of molecules inside the stretched gel.²⁰ The size of the RQCs varies with the amount of strain and is not constant over the gel body unless the gel is equilibrated for a prolonged period of time.^13,15 Slices 1 to 5 are located outside the polymer and hence only a singlet is observed for lithiated PMDTA in this region.

Fig. 2 (top) The reaction between nBuLi and PMDTA yields lithiated PMDTA via the intermediate [(nBuLi)₂PMDTA]₂. (bottom left) Slice-selective ⁷Li NMR spectra (slices 1 to 19) of the reaction within a polystyrene gel, 3 days after the addition of PMDTA. (bottom right) Slice-selective ¹H NMR spectra (slices 11 to 19) of the same sample.

Increasingly broad signals in slices 17–13 with chemical shifts between 2.3 and 1.6 ppm mark the reaction front between PMDTA and nBuLi, at which multiple dynamic processes take place.²¹ Although the corresponding ¹H spectra are relatively crowded with signals of different reaction components as well as residual signals from the polymer, the isolated region of the nBuLi α-CH₂ protons (−0.5 to −1 ppm) is quite informative (Fig. 2 bottom right): the signal for the nBuLi hexamer^22,23 at −0.92 ppm moves downfield and broadens from slice 19 to 13, presumably due to unspecific deaggregation and/or complexation of nBuLi by PMDTA. These processes are fast to intermediate on the NMR timescale such that only averaged chemical shifts are observed by ¹H and ⁷Li NMR. From slice 12 onwards, the α-CH₂ region is free of signals indicating that nBuLi has completely reacted to butane.

The most remarkable feature in slices 18–13 is the appearance of an additional, unshifted ¹H resonance at −0.59 ppm with a maximum intensity in slice 15. This signal was assigned to the complex [(nBuLi)₂PMDTA]₂ (see Fig. 2 top), which seems to be remarkably stable and has been characterised by X-ray crystallography, ¹H NMR and quantum chemical calculations as an intermediate in the lithiation of PMDTA before.²¹ Further ¹H signals reported for [(nBuLi)₂PMDTA]₂ were found in the crowded regions of the corresponding slices (see ESI†).

The connection between the reaction progress and the appearance of the peak at −0.59 ppm can nicely be illustrated by integrating this peak and the remaining nBuLi α-CH₂ signal over the course of three days. Fig. 3 illustrates the motion of the reaction front, where the nBuLi α-CH₂ signal (orange) vanishes and the signal of the corresponding CH₂-moiety within the [(nBuLi)₂PMDTA]₂ complex (green) appears. The latter builds up and decays exclusively at the reaction front, indicating its intermediate character. While 3 h after the addition the front is relatively sharp with little formation of [(nBuLi)₂PMDTA]₂, it becomes significantly blurred as it moves downwards with time. This is in accordance with what would be expected from diffusion in a gel.²⁴ The dip in the nBuLi concentration which is initially observed in the centre of the gel most likely arises from slow and incomplete diffusion of nBuLi into the polymer during sample preparation.¹³ This again underlines that the nBuLi hexamer (M_w = 383 g mol⁻¹) may be regarded as rather static compared to the PMDTA molecules (M_w = 173 g mol⁻¹). No separate ⁷Li NMR signal was found for [(nBuLi)₂PMDTA]₂, most likely due to fast dynamic exchange with the signal of nBuLi. Likewise, no evidence was found for further breaking down of [(nBuLi)₂PMDTA]₂ into smaller units representing active species/intermediates during the lithiation of PMDTA. Note that a monomeric nBuLi·PMDTA adduct has been proposed but its existence has not yet been experimentally confirmed.^25,26

Fig. 3 Relative integrals for the ¹H NMR signals at −0.59 ppm (green) and that of the nBuLi α-CH₂ protons (orange) as function of the slice number, 3 h, 1, 2 and 3 days after addition of PMDTA to nBuLi inside the pre-swollen polystyrene gel. All signals high-field to −0.59 ppm were attributed to “nBuLi”.

In the present study we could show that slice-selective NMR spectroscopy is a simple method to perform single-shot NMR titrations and in situ observation of reactions, using a routine NMR instrument. The “fast titration” was successfully tested in the complexation of a lithium salt by 12-crown-4, where it was able to reproduce a conventional ⁷Li chemical shift titration curve. Reaction monitoring by slice-selective NMR was tested in the reaction between PMDTA and nBuLi, where the previously characterised intermediate [(nBuLi)₂PMDTA]₂ could be identified. A stretched polystyrene gel was used as medium (i) to slow down the reaction and avoid convection, (ii) to immobilise one reactant (nBuLi) with respect to the other (PMDTA) and (iii) to principally enable also the observation of ⁷Li RQCs as additional source of structural information. Polystyrene is chemically inert, tolerates highly reactive reagents,²⁰ and is swollen by a broad range of solvents.²⁷ A further advantage of the gel method is the possibility to adjust the slope of the reaction front and hence to “zoom” into the interesting region. It should therefore be possible to apply slice-selective NMR to a broad range of reactions to obtain information about mechanisms as well as stoichiometry of complexes and products.

T. N. thanks the Studienstiftung des Deutschen Volkes for financial support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Detailed procedures, spectra, additional schemes. See DOI: 10.1039/c4cc08329f

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