Gradient-based pulse sequences for benchtop NMR spectroscopy

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spectroscopy

Jonathan Farjon, Boris Gouilleux, Patrick Giraudeau

To cite this version:

Jonathan Farjon, Boris Gouilleux, Patrick Giraudeau. Gradient-based pulse sequences for benchtop

NMR spectroscopy. Journal of Magnetic Resonance, Elsevier, 2020, 319, �10.1016/j.jmr.2020.106810�.

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1

3

Gradient-based pulse sequences for benchtop NMR spectroscopy

4

5

6

Boris Gouilleux

a

, Jonathan Farjon

b

, Patrick Giraudeau

b,⇑ 7 a

Université Paris-Saclay, ICMMO, UMR CNRS 8182, RMN en Milieu Orienté, France 8 b

Université de Nantes, CNRS, CEISAM UMR 6230, F-44000 Nantes, France 9 10 11 1 3

a r t i c l e i n f o

14 Article history: 15 Received 26 June 2020 16 Revised 12 August 2020 17 Accepted 13 August 2020 18 Available online xxxx 19 Keywords: 20 NMR spectroscopy 21 Benchtop 22 Solvent suppression 23 Pure-shift 24 Ultrafast 25 Monitoring 26 Authentication 27 2 8

a b s t r a c t

29

Benchtop NMR spectroscopy has been on the rise for the last decade, by bringing high-resolution NMR in

30

environments that are not easily compatible with high-field NMR. Benchtop spectrometers are accessible,

31

low cost and show an impressive performance in terms of sensitivity with respect to the relatively low

32

associated magnetic field (40–100 MHz). However, their application is limited by the strong and

ubiqui-33

tous peak overlaps arising from the complex mixtures which are often targeted, often characterized by a

34

great diversity of concentrations and by strong signals from non-deuterated solvents. Such limitations

35

can be addressed by pulse sequences making clever use of magnetic field gradient pulses, capable of

per-36

forming efficient coherence selection or encoding chemical shift or diffusion information. Gradients

37

pulses are well-known ingredients of high-field pulse sequence recipes, but were only recently made

38

available on benchtop spectrometers, thanks to the introduction of gradient coils in 2015. This article

39

reviews the recent methodological advances making use of gradient pulses on benchtop spectrometers

40

and the applications stemming from these developments. Particular focus is made on solvent suppression

41

schemes, diffusion-encoded, and spatially-encoded experiments, while discussing both methodological

42

advances and subsequent applications. We eventually discuss the exciting development and application

43

perspectives that result from such advances.

44

45 46 47

48 1. Introduction

49 Making an analytical technique accessible to non-experts at a

50 reasonable cost is the key towards its widespread use. For decades,

51 methods such as mass spectrometry, near infrared or Raman

spec-52 troscopies have made their way towards food industries, hospitals,

53 and even airports or crime scenes[1–3]. NMR has also reached

54 such an early level of accessibility through portable relaxometry

55 equipment [4–6]. However, until the 2010s, NMR spectroscopy

56 remained the privilege of relatively big academic laboratories or

57 industries that could afford purchasing a high-field spectrometer

58 and keeping it in good operating conditions thanks to specialized

59 staff. This paradigm started to change in the early 2010s thanks

60 to pioneers such as Blümich and co-workers, who designed the

61 first benchtop NMR spectrometer, based on a fist-sized magnet

62 capable of being placed under a fume hood while providing a

suf-63 ficient homogeneity to yield NMR spectra with decent quality[7].

64 Within a few years only, several commercial companies have built

65 their success on the development of portable NMR spectrometers,

66 and hundreds of such portable devices have been installed in

67

industries or labs who were not necessarily familiar with

high-68

field NMR. Benchtop NMR spectrometers, whose magnetic field

69

range from 1 to 2.3 T, are capable of detecting all common nuclei

70

(in particular1_{H and}13_{C) with an excellent stability and an}

impres-71

sive linewidth (<0.5 Hz)[8].

72

Benchtop NMR spectroscopy is not destined to replace

high-73

field NMR, but rather to act as a complement in situations where

74

high-field magnets are not accessible or too expensive. This

75

includes quality control at production sites in food or

pharmaceu-76

tical industry[9,10], as well as the online monitoring or control of

77

chemical reactions[11] or biological processes[12]. In all these

78

applications, targeted samples consist of very complex mixtures

79

with a broad diversity of compounds in terms of molecular

struc-80

ture and concentration. Such complex mixtures appear rather

81

incompatible, however, with the intrinsic limitations of medium

82

field instruments, ie. a reduced sensitivity and ubiquitous peak

83

overlap arising from the limited spectral width in frequency units.

84

These limitations make benchtop NMR ill-suited to resolve

individ-85

ual components within a complex mixture, especially when the

86

peaks from compounds of interest are overlapped with those of

87

more concentrated compounds (such as non-deuterated solvents

88

in the case of reacting mixtures).

https://doi.org/10.1016/j.jmr.2020.106810

⇑ Corresponding author.

E-mail address:patrick.giraudeau@univ-nantes.fr(P. Giraudeau).

Journal of Magnetic Resonance xxx (xxxx) xxx

Contents lists available atScienceDirect

Journal of Magnetic Resonance

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j m r

Please cite this article as: B. Gouilleux, J. Farjon and P. Giraudeau, Gradient-based pulse sequences for benchtop NMR spectroscopy, Journal of Magnetic

Uncorrected

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89 Such overlap and dynamic range issues also exist -to a lesser

90 extent- at high magnetic field, where a whole galaxy of innovative

91 methods have been developed to deal with them. Common

high-92 field NMR methodologies for the analysis of mixtures include

93 advanced pulse methodologies, such as fast multi-dimensional

94 methods[13], diffusion-ordered spectroscopy (DOSY)[14],

homod-95 ecoupled ‘‘pure-shift” acquisitions[15], and a large toolbox of

sol-96 vent signal suppression methods[16]. All these pulse sequences,

97 which are now part of the daily arsenal of high-field NMR

spectro-98 scopists, share the common feature that they require robust -and

99 sometimes strong- pulsed magnetic field gradients (PFGs), making

100 use of the gradient coil which is now present in most of high-field

101 liquid-state NMR probes. In 2020, high-field NMR experts would

102 probably not even imagine developing a new pulse sequence

with-103 out a clever use of PFGs. Therefore, it is relatively straightforward

104 to imagine how PFGs could help maximizing the potential of

105 benchtop NMR spectroscopy, by making it possible to implement

106 and optimize pulse sequences that were initially designed for the

107 analysis of complex mixtures at high field.

108 The idea of implementing PFGs on a benchtop spectrometer

109 started from a discussion at Euromar 2013 in Crete with Stefano

110 Caldarelli, a true visionary in the world of complex mixture NMR,

111 who unfortunately left us prematurely in 2018. It took a couple

112 of years -and passionate discussions- until a manufacturer

(Magri-113 tek) was able to implement a gradient coil on its 43 MHz1_H/19_F

114 spectrometer, whose specifications were thought to accommodate

115 the strong requirements of echo-planar spectroscopic imaging

116 trains such as those included in ultrafast 2D pulse sequences

117 [17,18]. This was a rather impressive technical achievement

con-118 sidering the strong space limitations associated with benchtop

119 spectrometers. Until 2015, gradient pulses could only be included

120 in benchtop NMR pulse sequences by making use of shim coils.

121 Such feature was already sufficient to implement gradient-based

122 coherence selection in multi-pulse experiments, but was relatively

123 limited in terms of gradient strength and robustness required to

124 implement more advanced experiments. After 2015,

gradient-125 based experiments such as solvent-suppression schemes [19],

126 spatially-encoded pulse sequences enabling ultrafast 2D or

pure-127 shift acquisitions[20], and DOSY[21], could be successfully

imple-128 mented thanks to the gradient coil and to the pulse programming

129

capabilities of benchtop spectrometers (Fig. 1). By significantly

130

improving the ability of benchtop NMR to separate spectra from

131

mixture components, these approaches already opened up new

132

application perspectives, in the fields of reaction and process

mon-133

itoring, as well as for authentication and quality control.

134

This article aims at reviewing the recent methodological

135

advances making use of PFGs on benchtop spectrometers and the

136

applications that resulted from these developments. Note that

137

basic coherence-selection gradient schemes -that can be

per-138

formed through shim coils on any benchtop spectrometer- have

139

been excluded from the scope of this paper. The article is split into

140

three main parts that successively address solvent suppression

141

schemes, DOSY, and spatially-encoded experiments, while

dis-142

cussing both methodological advances and subsequent

applica-143

tions in each part. We eventually discuss the exciting

144

development and application perspectives that result from such

145

advances, since gradient-based benchtop NMR spectroscopy is still

146

in its early days.

147

2. Solvent elimination schemes

148

2.1. Methods

149

At medium field, the detection of 1H signals from analytes of

150

interest is hampered by overlap with intense signals coming from

151

protonated solvents, and also due to the dynamic range of the

152

receiver which is optimized for the most intense signals. In the

153

field of chemical synthesis, the use of solvent mixtures makes this

154

issue even more critical. In the analysis of biologically relevant

sys-155

tems, the strong water signal is also a major issue. In this context,

156

efficient solvent elimination schemes become essential to improve

157

the detection of relevant analytes.

158

Different schemes able to suppress the solvent peaks, either

159

before or after the excitation, have already been explored at high

160

magnetic fields. The main criteria to define their efficiency include

161

i) the ability to decrease the intensity of the solvent signal(s), ii) the

162

selectivity of solvent suppression and iii) the analytical features

163

such as repeatability and robustness[16]. Meeting these criteria

164

is made difficult by the radiation damping effects [22] on the

Fig. 1. The implementation of a gradient coil in a benchtop NMR spectrometer has allowed the development of advanced pulse sequences such as gradient-based solvent suppression (here, the WET pulse sequence is shown), diffusion-ordered spectroscopy, or spatially-encoded experiments (here, the ultrafast COSY experiment is shown). These new developments have opened new applications for benchtop NMR spectroscopy of complex mixtures. Note that the B0gradient is orthogonal to the flow, a particular

feature of benchtop spectrometers that facilitates the implementation of spatially-encoded experiments under flow conditions.

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165 strong solvent signals, and by the faraway solvent contribution

166 arising from spins located on the edge of the rf coil and

contribut-167 ing to the broad solvent peak tails. The weight of such faraway

sol-168 vent contribution is related to the inhomogeneity of the magnetic

169 field, making this effect hardware-dependent[23]. The design of

170 solvent suppression schemes should fight against these effects,

171 particularly trying to avoid baseline distortions that would be

172 detrimental to detect the peaks of interest. While such effects have

173 been extensively studied at high field, benchtop NMR comes with

174 its own specificities as regards solvent suppression. While faraway

175 solvent effects are still present, radiation damping is much less of a

176 concern compared to high-field NMR. However, the spectral range

177 is much lower, making selectivity issues the main difficulty in

178 implementing solvent suppression schemes. Additional difficulties

179 can arise from the13_{C satellites of some solvents that can overlap}

180 with peaks of interest. Finally, pulse sequences that are not too

181 long are required, to be compatible with flow effects in the case

182 of flowing samples.

183 At high field, the simplest and most popular solvent

suppres-184 sion scheme is the continuous wave presaturation (Sat)[24]. On

185 a benchtop spectrometer, Sat was used, for instance, to remove

186 the water signal of urine samples in the context of a metabolomics

187 study on type 2 diabete[25]. However, Sat is not the most robust

188 scheme and is not compatible with flow conditions, due to the long

189 saturation delay. Moreover, a long presaturation hampers the

190 detection of protons in exchange with the solvent making a

com-191 prehensive analysis difficult, especially in the case of biological

192 samples. In this context, gradient-based solvent elimination

193 schemes are much more promising, and thanks to the

implementa-194 tion of a gradient coil, most popular gradient-based pulse

195 sequences were implemented on a benchtop spectrometer[19].

196 Examples of such pulse sequences are shown inFig. 2, highlighting

197 those which have been the most widely applied at medium field so

198 far. These pulse sequences correspond to two very different

199 approaches to remove the unwanted solvent peaks, both relying

200 on a clever use of gradient pulses. WET-180-NOESY (Fig. 2A)

selec-201 tively defocuses the solvent magnetization while leaving those of

202 relevant analytes unaffected before excitation, while WATERGATE

203 W5 (Fig. 2B) selectively refocuses all magnetizations except those

204 from user-defined regions that contain the solvent peaks. Both

205 methods present their own advantages and drawbacks, which are

206 discussed below.

207 In the case of small molecules, the efficiency of various solvent

208 suppression schemes at 43 MHz was compared by Gouilleux et al.

209 [19], both under static or flow conditions, in non-deuterated water.

210 Fig. 2C shows such comparison in the case of lactate, which

repre-211 sents a challenging case in terms of selectivity since the quadruplet

212 of lactate is only 30 Hz away from water at 43 MHz. As shown in

213 Fig. 2C, Sat is not suitable to efficiently remove the water signal

214 and suffers from a lack of selectivity. The WET pulse sequence

215 [26]is more selective but the baseline remains affected by faraway

216 water. Variants of the WET pulse sequence are more immune to

217 faraway water effects like WET-CP and WET-180[27]. They lead

218 to a fully resolved quadruplet of the lactate CH signal. A flat

base-219 line is also obtained when a NOESY 1D scheme [28] is

imple-220 mented. Selective refocusing methods such as WATERGATE[29]

221 or excitation-sculpting (ES) [30] are less efficient in this case

222 because -although the solvent signal is efficiently suppressed-

J-223 modulation occurs during the pulse sequence and affects the

mul-224 tiplet lineshapes and relative intensities. Such effects also exist at

225 high field but are less critical because spectra are not dominated

226 by J-coupling. Overall, the WET-180-NOESY was found to be

opti-227 mal to preserve the quantitative performance, both in terms of

228 trueness of integral ratios and repeatability. It was also shown to

229 remain the most competitive pulse sequence for the detection of

230 small molecule signals in flow conditions (up to 2.0 mL.min1).

231

In the example discussed above, selectivity was the main issue

232

but sensitivity was not critical since the analyte (lactacte) was

con-233

centrated at 0.2 mol.L1. When concentrations are lower, one may

234

rely on pulse sequences which are less selective but more efficient

235

in removing the water signal. Bouillaud et al. evaluated several

236

signal-suppression approaches to detect lipids in water solutions,

237

on the example of lipid-enriched microalgae cells studied under

238

flow conditions [31]. Selectivity is much less challenging here

239

(the CH2lipid peak that was targeted was 150 Hz away from the

240

water signal) but lipid concentrations can be relatively low. In this

241

case, WET-180-NOESY was found inefficient to reduce the water

242

signal at a level that would enable the detection of the lipid peak.

243

Better results were obtained with the WATERGATE-W5 pulse

244

sequence, allowing a reduction by 27,000 of the water signal.

245

While this pulse sequence leads to J-modulation effects, this was

246

not critical here since the region of interest was a complex and

247

broad fingerprint rather than a well-resolved signal.

248

These examples show that gradient-based solvent-suppression

249

pulse sequences offer complementary tools to remove solvent

sig-250

nals. These methods were already implemented in different

251

domains of applications, from synthesis reaction to bioprocess

252

monitoring, as detailed in the next paragraphs.

253

2.2. Applications

254

Promising applications of benchtop NMR spectroscopy arise

255

from their combination with flow chemistry[11,32,33]. Benchtop

256

NMR can be implemented in line with the flow reactor, offering

257

an opportunity to better optimize and control conversion rates or

258

selectivities. In this context, solvent suppression is a major issue,

259

and the gradient-based pulse sequences described above have

260

been used in different studies.

261

In 2017, Legros et al. used benchtop NMR as an inline detector

262

to monitor the neutralization of mustard gas mimetics through a

263

portable flow chemistry setting[34]. Two solvent signals needed

264

to be suppressed, which was achieved thanks to a

WET-180-265

NOESY pulse sequence combined with a Sat block (Fig. 3A,B).

266

Thanks to this method, signals from the starting (CEES) and

neu-267

tralized (CEESO) chemicals involved in this flow-depolluting

pro-268

cess could be quantified, making it possible to efficiently monitor

269

the conversion yield.

270

The flow chemistry community is also very active in developing

271

autonomous self-optimizing reactors that combine a flow reactor,

272

an inline analytical detector and an algorithm that can integrate

273

results from inline analysis and update reaction parameters in real

274

time. Benchtop NMR is particularly attractive in such an intelligent

275

flow chemistry setting, and Cronin et al. performed pioneering

276

work in this field, demonstrating that benchtop NMR could be a

277

powerful technology for the optimization of an imine synthesis

278

from 4-fluorobenzaldehyde and aniline, using trifluoroacetic acid

279

as a catalyst[32]. The performance of such approach would

cer-280

tainly benefit from advanced solvent suppression methods. Along

281

this line, Cortés-Borda et al. showed that inline benchtop NMR

282

including WET-180-NOESY solvent suppression could be used to

283

optimize the yield of a critical step in the flow synthesis of a

natu-284

ral product, carpanone[35](Fig. 3C).

285

Another rapidly expanding application field for benchtop NMR

286

is the real-time monitoring and control of bioprocesses. Indeed,

287

bioprocesses involve complex and multiple molecular

transforma-288

tions for which NMR spectroscopy would be an ideal technique,

289

but these processes often take place in environments which are

290

not compatible with high-field NMR. NMR relaxometry and MRI

291

were historically the first approaches to monitor

bio-292

transformations in foods or cells [12]. With the development of

293

benchtop spectrometers, one pioneering application by Blümich

294

and co-workers was to follow fermentation by detecting and

quan-B. Gouilleux et al. / Journal of Magnetic Resonance xxx (xxxx) xxx 3

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295 tifying metabolites coming from the degradation of glycerol and

296 glucose by microbial systems [36]. Authors were able to detect

297 metabolites and access kinetic information despite the huge water

298 signal. However, when analyte concentrations become lower and/

299 or when targeted peaks are close to the solvent signal, bioprocess

300 monitoring by NMR spectroscopy can benefit from efficient solvent

301 suppression methods. Soyler et al. demonstrated how

WET-180-302 NOESY could be used to quantitatively monitor the enzymatic

303 hydrolysis of sucrose -an important reaction in food industry- by

304 integrating peaks that were less than 40 Hz away from the water

305 signal[37]. A different approach was taken by Bouillaud et al. to

306 monitor intracellular microalgal lipids biosynthesized during a

307 microalgae culture, where water represented more than 95% of

308 the sample volume and prevented the detection of smaller lipidic

309 signals. With the help of the WATERGATE W5 pulse sequence

310 (Fig. 2B), it was possible to assess the relative amount of lipids

311 for different microalgae starving states, from starved to semi

312 starved and non-starved cultures[31]. To go ahead with the

mon-313 itoring of living microalgae in physiological conditions, a

photo-314 bioreactor was coupled to a benchtop NMR apparatus [38]

315 (Fig. 4A). It was possible for the first time to follow the lipidic

accu-316 mulation in real time in vivo for 2 weeks during the course of a

317 microalgal nitrogen starvation period thanks to the acquisition of

318 one W5 1D spectrum per hour (Fig. 4B). Thus, the overall lipid

sig-319

nal at 1.2 ppm was used to the online evaluation of the lipidic

con-320

tent. The NMR lipidic monitoring data were in perfect agreement

321

with the offline-based GC data with a correlation factor R2_higher

322

than 0.999. Benchtop NMR with WATERGATE W5 was able to

323

detect lipidic intracellular concentrations at 9 mg.L1and to

quan-324

tify lipids from 30 mg.L1.

325

3. Diffusion-ordered spectroscopy

326

3.1. Methods

327

Diffusion NMR is a central analytical tool used in various fields

328

of applications such as the analysis of complex mixtures, the study

329

of dynamic phenomena, the measurement of hydration radii or

330

even the study of emulsion-like samples[39,40]. This methodology

331

– nowadays readily available on high-field spectrometers – is

332

really promising in the context of benchtop NMR to further extend

333

the scope of applications. Diffusion NMR at low magnetic field is

334

not a brand new idea. Many interesting works have been reported

335

early with the measure of bulk diffusion coefficients (D) of samples

336

to get dynamic insights. Here, we focus on the very recent ability of

337

gradient-equipped benchtop spectrometers to perform diffusion

338

NMR with sub-ppm-chemical shift resolution, corresponding to

x

y

_{y y}

180°

90°

A

B

C

Fig. 2. Most efficient gradient-based solvent suppression pulse sequences on a benchtop spectrometer; (A) WET-180-NOESY and (B) WATERGATE W5. (C) Comparison of the performance of different solvent suppression methods at 43 MHz, illustrated on the example of a lactacte sample in H2O.Fig. 2C reproduced with permission from[19]. Sat:

presaturation; CP: composite pulse; WET: water suppression enhanced by T1effects; ES: excitation-sculpting; Sel: selective; PE: perfect-echo.

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339 the so-called diffusion ordered spectroscopy (DOSY). To discuss the

340 potential, but also the challenges of DOSY applied at low magnetic

341 field, the basic features of approach are shortly described. In DOSY,

342 a series of NMR experiments – based on PFGSTE (pulsed-gradient

343 stimulated echo) or PFGSE (pulsed-gradient spin echo) – is carried

344 out with incremented values of the gradient amplitude. In the

345 absence of convection and for unrestricted diffusion, the signal

346 amplitude decays as the gradient amplitude increases at a rate

347 described by the Stejskal-Tanner equation[39]:

348

S¼ S0eDðcgdÞ

2_D

350 350

351 where S0is the signal observed in the absence of a gradient, D is the

352 translational diffusion coefficient of the considered compound,

c

is

353 the gyromagnetic ratio, d is the gradient duration,Dis the diffusion

354 time and g corresponds to the amplitude of the pulse-field-gradient.

355 This signal attenuation is common for all the spins belonging to a

356 given molecule that exhibits a purely translational diffusion

behav-357 ior. In practice, a DOSY map is usually obtained by fitting the signal

358 decay to the Stejskal-Tanner equation above. This processing results

359 in 2D-peaks with a Gaussian shape along the diffusion dimension

360 whose width depends on the quality of the fit, which is particularly

361

critical when peaks are overlapped such as in complex mixtures. To

362

deal with this limitation, the processing of DOSY data has been the

363

subject of numerous multivariate signal processing developments,

364

notably by the group of Nilsson and co-workers[41,42].

365

In principle the progress in acquisition and processing methods

366

that DOSY has undergone in the last two decades can be

trans-367

ferred on modern benchtop NMR spectrometers equipped with a

368

gradient coil. However, it must be kept in mind that the reliability

369

of the DOSY processing highly depends on the quality of the input

370

data in a far more critical way than in 2D FT-NMR. In this respect,

371

the inherent reduction of sensitivity and resolution associated with

372

benchtop NMR may be detrimental for the DOSY processing,

373

resulting in 2D maps with a misleading and unsatisfactory

diffu-374

sion dimension. Thus, a special emphasis in the choice of the

375

pulse-sequence and the data treatment should be paid to tackle

376

such pitfalls. In spite of a SNR (signal-to-noise ratio) reduction of

377

p

2, pulse-sequences based on PFGSTE are highly recommended

378

to avoid signal distortions occurring with PFGSE due to inherent

379

J-modulation. This well-established consideration is all the more

380

important in benchtop NMR where strong couplings are frequently

381

present. Assemat et al. showed that the combination of bipolar

382

PFGSTE with a LED (longitudinal eddy current delay) block

pro-383

vides –on a 43 MHz apparatus– diffusion-weighted spectra with

384

minimal line-shape and baseline distortions, even at 70% of the

385

maximal gradient amplitude [21]. Following the acquisition of

386

the data, two different types of DOSY processing are nowadays

387

available which are classified as univariate and multivariate. The

388

univariate processing corresponds to a fitting procedure as

men-Fig. 3. A and B) 43 MHz spectra recorded on a reacting mixture involved in the neutralization of a mustard gas mimetic in flowing conditions (3 mL.min1_{). A)}

Conventional 1D spectrum. The asterisks * match with the13

C satellites lines arising from the methanol resonance and MSA refers to methane sulfonic acid. The signals of interest are severely overlapped by the huge methanol signal at 3.3 ppm B) low-field spectrum on the same mixture obtained with the WET-180-NOESY experi-ment. Thanks to this pulse sequence, the CEESO peak at 3.8 ppm can be efficiently detected through the flow reaction even at high flow-rate. Readers are referred to Ref.[34]for detailed peak assignments. C) Autonomous self-optimizable flow reactor used for the optimization of the derivative 8 and relying on benchtop NMR as an inline detector. Red dashed lines put into relief units under the control of the algorithm. Figures A, B reproduced with permission from[34]; Figure C reproduced with permission from[35]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. A) Bioprocess system used for on-flow monitoring of the lipidic content within entire microalgae under nitrogen starving culture conditions. B) 3D stacked plot of1_{H WATERGATE W5 NMR spectra recorded during the cultivation. The}

residual water peak at 4.7 ppm and the significant growth of the main lipid peak at 1.2 ppm are highlighted. Figure adapted from[38]with permission.

B. Gouilleux et al. / Journal of Magnetic Resonance xxx (xxxx) xxx 5

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389 tioned above and relies on the assumption that the lines are

well-390 resolved along the spectral axis so that each signal attenuation

391 matches with a unique diffusion coefficient. In contrast, a

multi-392 variate processing deals with the whole spectrum at the same time

393 rather than treating signals separately. In this way, no assumptions

394 of the absence of overlaps is necessary whereas a prior knowledge

395 of the number of species involved in the mixture is highly

recom-396 mended. This choice between univariate or multivariate processing

397 deserves a specific attention in DOSY at medium field whereby

398 high-resolution conditions are not always fulfilled even for

rela-399 tively simple mixtures. A relevant choice depends in turn on the

400 situation and on the complexity of the 1D spectrum. As shown

401 by Assemat et al, in the absence of significant peak overlap, the

402 classical univariate processing is completely suitable and leads to

403 reliable DOSY maps with consistent D with the respect to the ones

404 measured with a HF spectrometer (Fig. 5A, B). The situation is more

405 nuanced as the degree of overlaps increases. D values become no

406 longer comparable with the reference values obtained with HF

407 DOSY (Fig. 5C, D). However, as seen inFig. 5C, medium-field DOSY

408 still separates most of the components of the pharmaceutical

for-409 mulation along the diffusion dimension and thus remains a

pre-410 cious support in the analysis of mixtures whose 1D spectrum is

411 overcrowded. Very recently, Mc Carney et al. proposed a solution

412 to deal with this case of severe peak-overlaps[43]. This approach

413 relies on a multivariate processing, e.g. SCORE algorithm, and was

414 evaluated on a mixture of two alcohols leading to highly

over-415 lapped signals. While the classical univariate process fails to

deli-416

ver a suitable DOSY map, SCORE gives consistent D with a clear

417

separation of the components involved in the mixture, as clearly

418

seen inFig. 6. Undoubtedly such a multivariate processing is a

rel-419

evant tool in the context of benchtop DOSY data and should further

420

extend the scope of mixtures analyzable with benchtop apparatus.

421

The aforementioned processing are available within the free GNAT

422

(General NMR analysis toolbox) software provided by the

Univer-423

sity of Manchester[42].

424

3.2. Potential applications

425

These pioneering results shed light on the potential of benchtop

426

DOSY in various fields of chemistry. The ability to deal with

sam-427

ples like real medicine formulations including analytes in a 10–

428

15 mM concentration range with a reasonable experiment

dura-429

tion (ca. 3 h), is quite promising for quality control purposes in a

430

pharmaceutical context. Furthermore, DOSY supported by an

431

adapted multivariate processing should counterbalance the lack

432

of resolution in the case of overcrowded spectra. Although no

433

applications have been reported yet, one could anticipate that

434

the analysis of reactive mixture in organic chemistry could benefit

435

from this approach. While the aforementioned solvent suppression

436

methods are efficient in most of the situations, benchtop NMR

437

remains somewhat unsuitable whenever a non-deuterated solvent

438

that exhibits a multi-lines pattern is used or in case of a mixture of

439

solvents. DOSY used here as a diffusion filter would enable the

440

analysis of reacting mixtures even in such challenging cases, as

Fig. 5. Examples of1

H DOSY maps recorded at 43 MHz on pharmaceutical samples and their HF counterparts. DOSY maps of a sample containing paracetamol (16 mM) and hypromellose 2910 (4000 cps) dissolved in DMSO d6recorded at 43 MHz (A) and at 500 MHz (B). DOSY maps obtained on a commercial sample of esomeprazole (Ranbaxy) in

DMSO d6at 43 MHz (C) and at 500 MHz (D). DOSY maps were obtained with a bipolar-PFGSTE-LED pulse-sequence and processed with a univariate processing whereby lines

automatically peak-picked were fitted to a mono-exponential decay. . Reproduced with permission from[21]

Uncorrected

(9)

441 suggested at high field by Esturau and Espinosa[45]. In the field of

442 forensics, this methodology may be a solution to identify

psy-443 choactive substances within a sample containing one of several

444 cutting-agents[46]. Finally, besides this opportunity to ease the

445 analysis of complex mixtures, these recent progress in benchtop

446 DOSY makes it attractive for other types of applications. One might

447 refer to the recent measurements of hydrodynamic radii of

poly-448 meric samples of dendrimers with chemical shift resolution[47].

449 Moreover, DOSY could provide valuable insights for the fine

char-450 acterization of emulsion-like samples involved in a wide range of

451 industrial fields such as food chemistry. Indeed, the accurate

mea-452 surement of droplet size in a liquid dispersion with chemical shift

453 resolution is highly desirable as highlighted earlier[48]. No doubt

454 that the recent demonstration of benchtop DOSY with sub-ppm

455 resolution will promote interest in this field of applications.

456 4. Spatially-encoded methods

457 4.1. Methods

458 Inspired from the world of magnetic resonance imaging (MRI),

459 spatially-encoded pulse sequences have highly contributed to

460 liquid-state spectroscopic pulse sequence developments at high

461 magnetic field for the last 20 years[18]. The combination of

mag-462 netic field gradients with frequency-selective or frequency-swept

463 pulses has made it possible to address sub-ensembles of spins in

464 a position-specific manner, opening the way to spatially encode

465 the spectroscopic information of interest. The pulse programming

466 capabilities of modern benchtop NMR spectrometers are

compati-467

ble with the design of such tailored pulses, and the introduction of

468

magnetic field gradients allowed their combination, resulting into

469

spatial encoding elements akin to those introduced earlier at high

470

field.

471

The first spatially-encoded pulse sequence which has been

472

introduced on a benchtop spectrometer is ultrafast (UF, or

single-473

scan) multi-dimensional NMR. This approach, introduced by Lucio

474

Frydman and co-workers in 2002, provides a spatially-encoded

475

alternative to the time-incremented approach that allows the

con-476

ventional acquisition of multi-dimensional spectra[49]. Its

princi-477

ple (Fig. 7) has been widely described in recent reviews[50]. The

478

pulse sequence has the same structure as conventional

multi-479

dimensional experiments (ie, for a 2D pulse sequence, preparation

480

- evolution (t1) - mixing - acquisition (t2)). However, the

conven-481

tional time-incremented period is replaced by a spatial encoding

482

period where a helicoidal magnetization winding is created by

483

the combination of linearly frequency-swept pulses with gradients.

484

During the course of acquisition, this information is decoded

485

through an echo-planar spectroscopic imaging (EPSI) scheme,

486

making it possible to record a complete 2D matrix in a single scan.

487

Ultrafast 2D NMR is probably the most general of fast

multi-488

dimensional methods, since it can be adapted to record any kind

489

of homo- or hetero-nuclear correlation. It suffers, however, from

490

sensitivity limitations associated with the large digital filter

band-491

width that need to be applied during acquisition due to the

fre-492

quency dispersion induced by gradients. UF NMR is also

493

associated with the need to compromise between spectral width

494

and resolution, although many pulse sequence optimizations have

495

been carried out to alleviate this compromise[51].

Fig. 6. Example of a diffusion experiment performed at 43 MHz and processed with a multivariate processing. The experiment was carried out on a mixture of 2-butanol and 1-octanol dissolved in DMSO d6. Spectral features of each component are extracted (left panel), together with the corresponding D values (right panel). The processing was

achieved by a SCORE algorithm while the input data were obtained with a Oneshot45 pulse-sequence[44]. Figure . reproduced with permission from[43]

Uncorrected

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496 At high magnetic field, UF 2D NMR has been widely applied to

497 solve a variety of challenges that were incompatible with

conven-498 tional 2D NMR such as the real-time monitoring of chemical

reac-499 tions on short timescales, the coupling with online

500 chromatography or with hyperpolarization [52]. Hybrid

multi-501 scan methods based on UF 2D NMR also provide an interesting

502 alternative to conventional 2D NMR for applications to

metabolo-503 mics and fluxomics, thanks to their high-throughput character

504 associated with an excellent repeatability[53]. Ultrafast 2D NMR

505 was the first gradient-based pulse sequence to be implemented

506 on a benchtop spectrometer in 2015[17]. Clean 2D COSY spectra

507 exhibiting lineshapes close to ideality could be obtained in a single

508 scan, highlighting the robustness of the gradient coil and amplifier

509 that were implemented on the benchtop system. Still, owing to the

510 low sensitivity of the benchtop spectrometer combined with the

511 sensitivity penalty associated with UF 2D NMR, a few transients

512 were preferred to yield an acceptable sensitivity (limit of detection

513 lower than 0.5 mol.L-1 reached in less than one minute of

experi-514 ment time)[54]. Such UF 2D COSY experiments were characterized

515 by an excellent repeatability (a few percent, as for their high-field

516 counterpart), opening promising application perspectives that are

517 described in the next paragraphs. Note that such a promising

per-518 formance was obtained with a maximal gradient strength of 16 G.

519 cm1on a 1 T apparatus. On a side note, it is interesting to cite the

520 work of Telkki and co-workers who implemented the ultrafast

521 methodology on a portable setting for ultrafast Laplace 2D NMR

522 [55]. Although out of the spectroscopy focus of the present article,

523 this work is highly interesting since it opens promising

perspec-524 tives for the characterization of liquid dynamics in heterogeneous

525 media.

526 Another very promising approach to simplify the analysis of

527 complex mixtures is pure-shift NMR, which simplifies the 1D or

528 2D spectra by removing the multiplicity arising from1_H–1_H

cou-529 plings in the1_{H dimension}_[15,56]_{. Methods based on the}

projec-530

tion of J-resolved 2D spectra were developed a long time ago,

531

albeit associated with a number of limitations in terms of

line-532

shapes[57]. More recently, pure-shift NMR has known a

renais-533

sance through the development of spatially-encoded methods

534

that combine selective, band-selective or chirp pulses with

gradi-535

ents so that coupling partners in a spin system are excited in a

536

position-specific fashion [58]. Such methods have become very

537

popular at high-field, but have only been implemented in 2019

538

on a benchtop spectrometer for the first time. Castaing-Cordier

539

et al. showed that a variety of popular pure-shift methods, such

540

as Zangger-Sterk or PSYCHE pulse sequences, could be efficiently

541

implemented and provided efficient broadband homonuclear

542

decoupling (Fig. 8)[20]. Pure-shift NMR spectra at 43 MHz suffered

543

from the same sensitivity limitation as their high-field counterpart

544

(i.e., only a few percent of the sensitivity of 1D experiments are

545

retained). In addition further methodological progress for this

546

technique could arise from the implementation of pulse sequences

547

that would be less sensitive to second order coupling effects. While

548

strong couplings are a known limitation of pure-shift NMR at high

549

field, this becomes even more critical at medium-field, where

550

strong coupling effects are ubiquitous, even for simple spin

sys-551

tems. Nevertheless, results reported on a variety of small- and

552

medium-sized molecular mixtures showed a nice resolution

553

improvement, as well as an encouraging repeatability. Applications

554

have not been yet described, but given the spectral simplification

555

capabilities of pure-shift NMR, one could envisage the same kind

556

of applications as UF 2D NMR (profiling, monitoring), focusing on

557

highly-concentrated or even hyperpolarized samples. Additional

558

perspectives could also come from pure-shift 2D heteronuclear

559

spectra[59,60], whose implementation on a benchtop

spectrome-560

ter was reported by Blümich and Singh[8]. Since they rely on a

nat-561

ural abundance 13_{C filter, such experiments do not require to}

562

compromise on sensitivity compared to conventional HSQC or

563

HMBC, and greatly simplify the 2D spectra of complex mixtures.

Fig. 7. (A) Ultrafast (UF) COSY pulse sequence and its application to (B) the real-time monitoring of a Heck-Matsuda Pd-catalyzed reaction. (C) Some of the 55 UF COSY spectra that were recorded every 3.6 min in the course of a 200 min reaction, monitored at 29°C with an initial concentration of 0.36 mol.L1_{for the limiting starting}

material. B and C . reproduced with permission from[17]

Uncorrected

(11)

564 A common feature of both UF and pure-shift methods is that

565 their spatially-encoded nature could appear incompatible with

566 flowing samples, making them ill-suited for monitoring

applica-567 tions that form one of the main applications of benchtop NMR.

For-568 tunately, in the spectrometers where these experiments were

569 implemented, the B0field and the associated gradient are

horizon-570 tal, owing to the design of the Hallbach magnets. Hence, the B0

gra-571 dient is orthogonal to the flow, and there is no impact of flow

572 specific to spatial encoding -just the regular effect of in-flow and

573 out-flow effects[61]. This explains why such experiments can be

574 readily applied to reaction monitoring (see next paragraph),

con-575 trary to their high-field counterparts that require specific pulse

576 sequence design to account for flow effects[62].

577 4.2. Application to reaction monitoring

578 Spatially-encoded methods have shown a high potential for

579 reaction monitoring at high-field thanks to their ability to simplify

580 overlapped spectra of complex mixtures. UF 2D NMR, in particular,

581 has been widely used for such applications, since it is compatible

582 with samples that evolve in the course of time while retaining

583 the ability of 2D NMR to separate overlapping peaks while

provid-584 ing information on chemical structures[63]. At high field, UF 2D

585 NMR has been used to provide real-time insight on organic

reac-586 tions such as the synthesis of pyrimidines[64]or the hydrolysis

587 of an acetal[65], but was also applied to the in situ monitoring of

588 electrochemical processes[66].

589 Thus, the implementation of UF 2D NMR on a benchtop

spec-590 trometer in 2015 opened immediate perspectives to disentangle

591 the heavily overlapped spectra obtained at 43 MHz. However, the

592 limited sensitivity of this method requires careful design of the

593 experimental conditions, so that analyte concentrations remain

594 above the limit of detection (typically 0.1 M). While benchtop UF

595 2D NMR is not suited to identify low-concentrated reaction

inter-596 mediates, it can be well suited to optimize reaction conditions in

597 a flow setting under the fume hood, which is not practical at high

598

field. In this context, Gouilleux et al. showed that UF COSY could be

599

used to monitor a Heck-Matsuda Pd -catalyzed reaction in a

non-600

deuterated solvent, within an NMR tube [17] (Fig. 7). Based on

601

55 UF spectra recorded in the course of a 200 min reactions, kinetic

602

parameters could be extracted, providing a convenient method to

603

optimize reaction conditions. 2D experiments were found crucial

604

in this context, since 1D NMR data failed to provide isolated peaks

605

for all the compounds of interest. In a follow-up study, the same

606

reaction was carried out in a flow setting, showing that the

reac-607

tion would perform much faster than in a tube thanks to the ability

608

to better control the reaction conditions[54], a conclusion

match-609

ing the one reached at high field by Foley et al.[67]. Mechanistic

610

details were deduced from the determination of conversion rates,

611

highlighting the potential of UF 2D NMR on a benchtop setting

612

for reaction monitoring.

613

4.3. Application to authentication

614

Another field where benchtop NMR has shown high promises is

615

the quality control or authentication of food products[68,69]. Most

616

food industries cannot afford a high-field equipment -that would

617

anyhow be incompatible with most food-production

618

environments- and need to rely on less informative analytical

619

methods (such as IR or Raman spectroscopies, or even NMR

relax-620

ometry) or to put the NMR analyses in the hands of external

com-621

panies. Benchtop NMR could be a game changer in this field, by

622

bringing NMR spectroscopy directly to food production and control

623

sites. In NMR-based food quality control, a ‘‘omics” approach is

624

generally taken [70], that consists in recording1_{H NMR}

finger-625

prints and processing them through statistical analyses to extract

626

relevant information from highly overlapped spectra -in particular

627

at medium field[68]. Data are compared to a database to provide

628

information on the geographical or botanical origin, organic

char-629

acter, possible adulteration, etc.

630

Since spectra are highly crowded, the potential of these

meth-631

ods could be significantly boosted by relying on acquisition

strate-632

gies that better separate peaks at the acquisition stage of the

633

analytical workflow. At high field, the potential of UF 2D and

634

pure-shift NMR approaches in ‘‘omics” workflows has been

635

demonstrated in a number of cases [71]. At medium field, this

636

potential is even higher since peak overlap is even more critical.

637

In addition, fast methods are needed to make the approach

high-638

throughput, towards routine quality control applications. In this

639

context, UF 2D NMR sounds like an ideal candidate to improve

640

the performance of benchtop NMR in a quality control context.

641

Such potential has been shown in 2016 by Gouilleux et al., who

642

relied on a fast (2.4 min) COSY experiment to assess the botanical

643

origin of edible oils (Fig. 9)[10]. They analyzed 23 edible oil

sam-644

ples from different origins, and they compared the fast 2D

645

approach with classical 1D NMR. For the same experiment time,

646

UF 2D NMR provided a much better separation between sample

647

groups, showing the input of 2D NMR as a tool to facilitate ‘‘omics”

648

benchtop analyses. In addition, they showed that this approach

649

could be used to build a PLS (partial least square) model capable

650

of detecting adulteration processes such as the addition of

hazel-651

nut oil into olive oil, a common fraud in food industry. While this

652

strategy offers a convenient approach to boost the control of food

653

products, it could also be applied to improve the performance of

654

other ‘‘omics” approaches which are emerging on benchtop

spec-655

trometers, such as point-of-care metabolomics[25].

656

5. Perspectives

657

Methodological advances at medium magnetic fields are

essen-658

tial to improve performances of benchtop NMR. In this context, the

Fig. 8. Pulse sequences for pure-shift1

H experiments with pseudo-2D acquisition mode. Double spin echo based methods: PSYCHE pulse sequence (A), Zangger-Sterk (ZS) pulse sequences with a Q3 (B) ReBURP (C) and rSNOB (D) 180° refocusing selective pulse. Example of a ZS spectrum obtained with a rSNOB pulse on a mixture of 8 metabolites (E) for JHHdecoupling as compared to the standard 1D1H spectrum

(F). Spectra were recorded in 28 min. Figure adapted from Ref.[20].

Uncorrected

(12)

659 recent examples described in this review highlight how

gradient-660 based methods are crucial to improve conventional techniques

661 by making it possible to implement recent pulse sequence

662 advances that were made at high field. These include solvent

elim-663 ination, DOSY and spatially-encoded schemes like ultrafast and

664 pure-shift experiments.

665 Further methodological advances could arise from the

combina-666 tion of several of these methods, in particular by combining solvent

667 suppression schemes with spatially-encoded methods. On the one

668 hand, for shift spectroscopy, designing a variety of 2D

pure-669 shift experiments, such as TOCSY-PSYCHE[72]or pure-shift HSQC

670 based on a BIRD filter[73], would offer better versatility. On the

671 other hand, a broader diversity of ultrafast 2D experiments could

672 be implemented -most likely those based on homonuclear

spec-673 troscopy for obvious sensitivity reasons. These include

multiple-674 quantum experiments, whose ultrafast version at high-field has

675

been shown to greatly simplify the analysis of complex mixtures

676

with overlapped peaks[74,75].

677

As discussed in this paper, DOSY also shows promising

perspec-678

tives at medium field, in particular for quality control applications.

679

Therefore, one could expect that the performance of DOSY will be

680

further improved by developments akin to those that were

681

recently made at high-field, in particular those aiming at

homode-682

coupling DOSY spectra such as ZS-DOSY[76], PSYCHE-DOSY[77]or

683

iDOSY PSYCHE[78]. For tracking fast changes within a complex

684

mixture, an ultimate advance could consist in the conjunction of

685

ultrafast schemes with DOSY methods. Such ultrafast DOSY

exper-686

iments have been successfully developed at higher fields[79]and

687

can record a 2D DOSY map in a fraction of a second.

688

Since all the aforementioned potential developments are based

689

on the incrementation of two or more dimensions, they could

ben-690

efit from non-uniform sampling to reach a better compromise

691

between sensitivity, resolution, and experiment time. For instance,

692

Kaziemierczuk et al. have shown the possibility to acquire

Pure-693

Shift spectra in 2 min, five time less than the conventional version

694

[80]. Implementing such strategies at medium field would

cer-695

tainly be beneficial to the quality of benchtop spectra.

696

Since a major application field of benchtop NMR focuses on

697

flowing samples, further developments should also consider

opti-698

mizing pulse sequences to minimize sensitivity losses arising from

699

flowing samples, that induce pulse-sequence specific signal losses

700

and distortions. While the configuration of benchtop NMR (with

701

the B0field and associated gradient orthogonal to the flow) is

rel-702

atively favorable compared to high-field, spatially-encoded

exper-703

iments would certainly benefit from the developments made at

704

high-field aiming at designing pulse sequences more immune

705

towards flow effects[62].

706

Still, one should keep in mind that spatially-encoded methods

707

come at a price to pay in terms of sensitivity. While many

applica-708

tions remain compatible with the sensitivity of such methods at

709

high-field, the limited sensitivity of benchtop NMR makes the

710

use of these pulse sequences limited to concentrated samples, thus

711

restricting the corresponding scope of applications. Therefore,

712

promising perspectives could arise from their combination with

713

hyperpolarization methods, in particular para-hydrogen-based

714

methods that are relatively cheap, general and portable, and can

715

be made compatible with flow configurations. Proof-of-concept

716

studies associating pH2 hyperpolarization with compact NMR

717

spectrometers for reaction monitoring have been published

718

[81,82], and a promising combination of SABRE -the

non-719

hydrogenative version of pH2- with benchtop NMR in a flow

chem-720

istry setting has been recently reported[83]. Dissolution-DNP

(d-721

DNP) [84] is also a hyperpolarization method that could boost

722

the potential of benchtop NMR. Current d-DNP hardware are not

723

easily compatible with the compact configuration of benchtop

724

NMR, but efforts towards transportable d-DNP hyperpolarized

725

agents could make it an interesting option[85].

726

The methodological developments described in this paper could

727

significantly widen the scope of applications of benchtop NMR

728

spectroscopy. Besides their beneficial contributions in reaction

729

monitoring and quality control, gradient-based methods could

730

rapidly take a central role in applications of growing interest in

731

chemistry, health sciences and forensics. The field of organic

chem-732

istry has witnessed the emergence of novel types of synthesis

plat-733

forms in the last few years. One might think of the recent

734

development of self-optimizing flow reactors[11,32] as well as

735

the reported organic synthesis robot driven by machine learning

736

algorithms in the sake of discovering new chemical reactivity

737

[86]. Within these modern platforms, benchtop NMR is recognized

738

as a valuable processing analytical tool. Nonetheless, the chemical

739

insight accessible by standard 1D experiments is quite limited in

740

the case of mixtures including several components, especially

Fig. 9. Illustration of the potential of 2D experiments for the profiling of food samples with benchtop NMR spectroscopy. (Top) Ultrafast 2D COSY spectrum recorded in 2.4 min on a sunflower oil sample in non-deuterated chloroform. (Middle) PCA analysis obtained with such UF 2D NMR experiments on 23 edible oil samples from different botanical origins. (Bottom) PCA on the same sample set with standard 1D experiments and a variable bucketing approach. Reproduced from[10]

with permission from Elsevier.