NMR methodology for complex mixture 'separation'

Article abstract of DOI:10.1039/c3cc48907h

Mixture 'separation' by NMR is demonstrated through the development of a pseudo 4D NMR experiment, 3D IPAP INEPT-INADEQUATE-HSQC, designed for the structural elucidation of ¹³C tagged compounds.

Full text of DOI:10.1039/c3cc48907h

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NMR methodology for complex mixture
‘separation’†
Nicholle G. A. Bell,a Lorna Murray,a Margaret C. Grahamb and Dusan Uhrın*a
Cite this: Chem. Commun., 2014,
50, 1694
ˇ
´
Received 21st November 2013,
Accepted 24th December 2013
DOI: 10.1039/c3cc48907h
www.rsc.org/chemcomm
Mixture ‘separation’ by NMR is demonstrated through the develop- nuclei. Once in place, the polarization transfer pathways are directed
ment of pseudo 4D NMR experiment, 3D IPAP INEPT- through these tags, reducing the complexity of spectra significantly.
INADEQUATE-HSQC, designed for the structural elucidation of As illustrated here, this approach has a potential to elucidate
a
13C tagged compounds.
molecular fragments of compounds contained in complex mixtures.
The bulk of HS is composed of oxygen rich HxCyOz compounds
The structural elucidation of compounds contained within complex with the Mw range of B200–1000 g molÀ1 10
Particularly prevalent
.
mixtures is a challenging task. Despite the advances in chromato- functionalities decorating aromatic and aliphatic skeletons of HS
graphy, some mixtures cannot be separated. Humic substances are the hydroxyl and carboxyl groups. These groups are crucial to the
(HS), produced by the biodegradation of plant and animal resi- interaction of HS with species such as heavy metals as well as the
dues,1 are the best known example of an ‘inseparable’ mixture self-association of HS molecules. The methodology presented here
consisting of thousands of organic compounds. Ubiquitous in aims at characterizing the aromatic moieties of HS carrying OH and
nature, they make up a considerable proportion of the Earth’s COOH groups and relies on introducing 13C-enriched –O13CH3 and
carbon pool and are key players in many biogeochemical processes. –COO13CH3 groups into HS compounds. HS have been methylated
In order to comprehend the functional roles of HS on a molecular in the past and the inspection of –O13CH3 resonances yielded some
level, their structural composition needs to be deciphered. So far HS rudimentary information about the nature of their COOH and OH
have been characterized only on the level of compound classes and groups.11 The novelty of our approach is that it uses labels to spy on
presence of individual functional groups.2–4 NMR spectroscopy and their neighbourhood, obtaining the 1H and 13C chemical shifts and
mass spectrometry are widely regarded as the two most promising 1H–1H and 1H–13C coupling constants of the nuclei in their vicinity.
analytical techniques for revealing the structure of individual HS
To achieve this aim we have designed a novel 3D NMR
molecules.5–7 Nevertheless, HS pose a considerable analytical chal- experiments, referred to here as 3D IPAP INEPT-INADEQUATE-
lenge to both techniques, e.g., the signal overlap in NMR spectra of HSQC (Fig. 1).
complex mixtures prevents separation of resonances belonging to
This NMR experiment can be viewed as a 3D extension of a 2D
individual molecules and hence their identification using standard INEPT-INADEQUATE.12 It is also related to 3D HCCH experi-
NMR techniques. Increasing the dimensionality of NMR experiments, ments.13,14 3D IPAP INEPT-INADEQUATE-HSQC exploits polarization
selective excitation, or DOSY spectroscopy8,9 are the three most transfer pathways shown in Fig. 1, and correlates the double-
common approaches attempted to circumvent these problems.
quantum (DQ) coherencies of long-range coupled carbons (F1) with
1
Unfortunately, these quickly fail when the complexity of mixtures corresponding single-quantum 13C chemical shifts (F2) and the H
2,3
increases. Inevitably, for chromatographically inseparable mixtures chemical shifts (F3). The polarizations transfer is tuned for
J
CC
the ‘‘separation’’ must be done spectroscopically. One way of (Fig. S1, ESI†) and correlates the chemical shifts of nuclei in
13
achieving this is by tagging the molecules with isotopically labelled 13CH3ÁÁÁ CHy (y = 0, 1) fragments. It starts and ends concurrently
on methoxy and aromatic protons located next to the methoxy
a EastChem, School of Chemistry, University of Edinburgh, King’s Buildings,
groups. For CH3ÁÁÁCH fragments it provides chemical shifts of all
West Mains Rd, Edinburgh, Scotland, EH9 3JJ, UK. E-mail: dusan.uhrin@ed.ac.uk
b School of Geosciences, University of Edinburgh, King’s Buildings, West Mains Rd,
Edinburgh, Scotland, EH9 3JN, UK
four nuclei and can therefore be regarded as a pseudo 4D experiment;
all three chemical shifts are obtained for the CH3ÁÁÁCq moieties. Since
the acquired NMR signal is filtered via isotopically enriched 13CH3
groups, the resulting spectra are significantly simplified.
† Electronic supplementary information (ESI) available: Materials and methods,
details of the optimization of NMR parameters, values of coupling constants, S/N
ratios, examples of processing of IPAP spectra and a suppression of signals from
aliphatic OMe groups. See DOI: 10.1039/c3cc48907h
The limited 1H and 13C chemical shifts range of methoxy groups
(Fig. S2, ESI†) necessitates the use of high digital resolution in the
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Fig. 1 The pulse sequence of the 3D IPAP INEPT-INADEQUATE-HSQC and its polarisation transfer pathways utilising various coupling constants.
The details of the pulse sequence are given in the ESI.†
F2 and F3 dimensions of the 3D spectra. In order to achieve this in a interpretation of the 3D IPAP INEPT-INADEQUATE-HSQC spectra.
realistic time, we have (i) optimized 1H and 13C carrier frequencies These can be eliminated by band-selective inversion of spins
and spectral widths in all dimensions, (ii) frequency shifted DQ resonating within the 65–95 and 15–45 ppm regions of the 13C
coherences during t1, (iii) aliased signals in the F2 dimension, (iv) spectra applied during the initial carbon spin-echo, 2D2, (see the
used non-uniform sampling and (v) acquired pure in phase and inset of Fig. 1). Spins resonating in these regions effectively receive a
2,3
antiphase (IPAP)15 13C-coupled multiplets during t3, as detailed in 3601 pulse, thus refocusing their
J
CC
couplings with the 13CH3O
the ESI,† Fig. S3–S5.
carbons and eliminating their signals (see Fig. S6, ESI†).
The proposed method is illustrated on a model mixture of
How sensitive is the 3D IPAP INEPT-INADEQUATE-HSQC experi-
nine benzoic acid derivatives, 1a–9a, which were fully methyl- ment? Because the DQ coherences are created between the fully
ated using 13CH3I, yielding compounds 1–9 (Scheme 1, average labelled 13CH3 carbons and natural abundance 13C spins, the
concentration of 1.4 mM).
theoretical sensitivity of this experiment is comparable to that of a
2D F2F3 (HSQC) (Fig. 2a and b) and 2D F1F3 (DQ) (Fig. 2c and d) refocused gradient-selected H, 13C HMBC optimized for JCH cou-
planes of the 3D IPAP INEPT-INADEQUATE-HSQC spectrum of the plings. We have determined the S/N ratios in 1D F3 traces extracted
model mixture illustrate the resolving power of this experiment. The from F1F3 planes of the 3D spectrum obtained by the addition of the
spectrum was analysed as outlined in Fig. 2e–g. Shown here are the 3D IP and AP INEPT-INADEQUATE-HSQC spectra. The values were
2D DQ (F1F3) planes extracted at the 13C shifts of the aromatic and normalized (Fig. S7, ESI†) to the average concentration of the
Me carbons (F2) indicated by red arrows in Fig. 2a and b. The methylated compounds (1.4 mM). Due to their singlet character,
obtained 13C and 1H chemical shifts allowed unambiguous identifi- the S/N is higher for –O13CH3 (60–1900 : 1) than for the aromatic
cation of the fragment highlighted in the inset. The fragment was resonances (57–600 : 1). Somewhat lower values were obtained for
assigned to the correct molecule by considering the electronic –COO13CH3 signals (40–240 : 1). These values clearly reflect the sizes
1
n
effects of OMe and COOMe groups on the 13C and H chemical of 2,3JCC couplings (Fig. S1, ESI†) involved in the polarisation transfer
1
shifts of benzene and by analysing the proton–proton and long- and can be increased for small coupling constants by optimizing the
range proton–carbon coupling constants determined in F3. The D2 intervals for smaller JCC couplings (6 Hz at present). This will have
inspection of the complete 3D spectrum lead to the identification of to be balanced against faster relaxation in larger molecules. We note
the fragments highlighted in Scheme 1 and their assignment to that the relaxation effects can be reduced by using 13CD2H in place of
individual molecules.
13CH3 groups. This modification opens a route toward a design of
When applied to HS, the signals from methylated aliphatic more efficient 3D INADEQUATE-based experiments thus compensat-
hydroxyl and carboxyl groups can potentially interfere with the ing partially for the loss of protons in 3CD2H groups.
Considering the lowest S/N observed (40 : 1), a 10-fold reduction of
the concentration would still yield spectra of adequate quality. Thus
assuming a 140 mM concentration of a 500 g molÀ1 Mw compound,
38.5 mg dissolved in 550 ml of CDCl3 are required per compound. A
19.3 mg strong mixture of compounds containing 500 unique sub-
stitution patterns by –OH and –COOH groups on aromatic rings will
therefore provide 3D spectra with sufficient S/N ratios to be analyzed. As
the methylation procedure includes extraction into the organic phase,
and thus selects a subset of HS compounds, simplification of these
complex mixtures is inherent to this process. This could be further
improved by employing partial fractionation of HS prior to methylation.
Scheme 1 Nine methylated compounds. The molecular fragments iden-
tified by the analysis of the 3D IPAP INEPT-INADEQUATE-HSQC spectrum
Obtaining 1H and 13C chemical shifts of the nuclei in the
based on the chemical shift and coupling constants are highlighted in bold.
vicinity OH and COOH by itself does not lead to a structure, and
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Fig. 2 2D projections of the 3D IPAP INEPT-INADEQUATE-HSQC spectrum obtained by the addition of the inphase and anti-phase 3D spectra. (a, b)
F2F3 HSQC and (c, d) F1F3 DQ projections of the aromatic (a, c) and methyl (b, d) regions of the spectrum. The circled signals belong to the aliased DQ
coherences of OMe carbons in compounds 4 and 5. See Fig. S4 (ESI†) for the explanation of their origin. Blue lines connect DQ coherences of CH3Á Á ÁCH
fragments detected simultaneously on the aromatic and methyl protons. The red arrow indicates the F2 coordinates of planes shown in (e–g). 2D DQ
(F1F3) planes extracted at the CH and OMe 13C chemical shifts indicated in (a and b) showing (e) aromatic, (f) ester and (g) methoxy proton regions of the
spectrum. The blue line indicates a shared DQ frequency. Observed splittings are annotated. (h) The identified molecular fragment.
such data needs to be interpreted by considering the precursors very promising approach to the analysis of complex mixtures
of HS molecules and their chemical shifts. However, it is clear such as HS. To the best of our knowledge, this work is the first
that this methodology supersedes information content of sim- example of a sophisticated NMR experiment designed to
ple 1H, 13C HSQC spectra of HS.16 The key is the increased explore the chemical environment around the tags, rather than
number of nuclei that are identified as belonging to the same just the signals from the tags themselves. This approach is not
molecule. Additional valuable information can be obtained by limited to methylation, other tags, containing NMR active, fully
hydrolysing the –COO13CH3 esters, while keeping the –O13CH3 abundant nuclei such as 15N or 19F can also be explored.
groups untouched. The resulting site-specific changes of Finally, many other mixtures can benefit from this approach,
chemical shifts of aromatic spins can be quantified through including those found in food, beverages, natural products, or
recording the 3D IPAP INEPT-INADEQUATE-HSQC of a partially biological samples, to name a few.
hydrolysed mixture (data not shown). Such information is very
This work was supported by the NERC grant NE/L00044X/1
valuable for the identification of fragments carrying both OH to MCG and DU. NGAB would like to acknowledge the support
and COOH groups.
of the University of Edinburgh Principal’s Career Development
In conclusion, we conjecture that tagging by 13C labelled Scholarship and NERC. We thank J. Bella for maintaining the
methyl groups in combination with 3D NMR spectroscopy is a NMR spectrometers.
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