13C NMR spectroscopy for the quantitative determination of compound ratios and polymer end groups

Article abstract of DOI:10.1021/ol403776k

¹³C NMR spectroscopic integration employing short relaxation delays was evaluated as a quantitative tool to obtain ratios of diastereomers, regioisomers, constitutional isomers, mixtures of unrelated compounds, peptoids, and sugars. The results were compared to established quantitative methods such as ¹H NMR spectroscopic integration, gas chromatography, and high-performance liquid chromatography and were found to be within <3.4% of ¹H NMR spectroscopic values (most examples give results within <2%). Acquisition of the spectra took 2-30 min on as little as 10 mg of sample, proving the general utility of the technique. The simple protocol was extended to include end group analysis of low molecular weight polymers, which afforded results in accordance with ¹H NMR spectroscopy and matrix-assisted laser desorption-ionization time-of-flight spectrometry.

Full text of DOI:10.1021/ol403776k

Letter
pubs.acs.org/OrgLett
13C NMR Spectroscopy for the Quantitative Determination of
Compound Ratios and Polymer End Groups
Douglas A. L. Otte, Dorothee E. Borchmann, Chin Lin, Marcus Weck,* and K. A. Woerpel*
Department of Chemistry, New York University, New York, New York 10003, United States
S
* Supporting Information
ABSTRACT: 13C NMR spectroscopic integration employing short
relaxation delays was evaluated as a quantitative tool to obtain ratios
of diastereomers, regioisomers, constitutional isomers, mixtures of
unrelated compounds, peptoids, and sugars. The results were
1
compared to established quantitative methods such as H NMR
spectroscopic integration, gas chromatography, and high-perform-
ance liquid chromatography and were found to be within <3.4% of
1H NMR spectroscopic values (most examples give results within
<2%). Acquisition of the spectra took 2−30 min on as little as 10 mg of sample, proving the general utility of the technique. The
simple protocol was extended to include end group analysis of low molecular weight polymers, which afforded results in
1
accordance with H NMR spectroscopy and matrix-assisted laser desorption-ionization time-of-flight spectrometry.
1
etermining product ratios is an important task in organic
Dchemistry because reactions often give mixtures of
products (e.g., diastereomers or regioisomers) that provide
information about the relative rates of formation and energies
of different products.1 1H NMR spectroscopy is particularly
useful, because single pulse experiments (or spectra obtained
using multiple scans with long delay times) give reliable
integrations that are directly related to compound ratios.2 In
some cases, however, peaks of two structurally related
400 MHz for H NMR spectroscopy. Generally, broadband
decoupled 13C NMR spectra that are suitable for compound
characterization can be used to obtain product ratios that are in
agreement with ratios determined by other methods.
The advantage of analysis by 13C NMR spectroscopy is
illustrated by the challenge of determining the diastereomeric
ratios of diols 1, 2, and 3 (Scheme 1). While the H NMR
1
Scheme 1. Diols 1, 2, and 3
1
compounds can be difficult to resolve by H NMR spectros-
copy.3 Other techniques, such as gas chromatography (GC)
and high-performance liquid chromatography (HPLC), provide
quantitative information, although the response factors of the
detection methods must be considered.4−6 Separation of
compounds by these techniques is not assured, and variation
of chromatography columns can be resource-intensive (in both
time and cost) and does not always lead to peak separation or
reliable ratios.7−9 13C NMR spectroscopy can be used as a
quantitative tool,10−14 but quantitative 13C NMR spectroscopy
requires long relaxation delays, which can result in long
acquisition times to achieve sufficient signal-to-noise ratios.
In this Letter, we demonstrate that proton-decoupled 13C
NMR spectroscopy using only short delays between pulses
provides a powerful technique for determining ratios between
small molecules and for performing polymer end group
analysis. Long delays are not required to obtain meaningful
ratios provided certain precautions are taken. The 20-fold
greater range of chemical shifts for 13C NMR spectroscopy
spectra of diols 1 and 2 are resolvable and give diastereomeric
ratios, the peaks assigned to the methyl groups at δ 1.5 ppm for
diol 3 cannot be integrated because of signal overlap (Spectrum
S-5 in the Supporting Information (SI)). GC of these similar
diols yielded inconclusive results: the peaks assigned to the two
diastereomers exhibited either no or poor resolution. In
contrast, several resonances of the diols 1, 2, and 3 (Spectrum
S-7 in the SI) were clearly resolved by 13C NMR spectroscopy.
The peaks could be integrated if spectra were acquired using
inverse-gated decoupling (IGD) pulse sequences with long
relaxation delays (D1) between pulses (30 s) to minimize
different nuclear Overhauser effects (NOEs) and longitudinal
relaxation times.10,12,15−17 The resulting experiments, however,
required several hours to obtain sufficient signal-to-noise that
allowed for the integration of spectra. By contrast, the same
product ratios could be obtained in minutes by integrating
spectra obtained by standard broadband decoupled (BBD)
compared to 1H NMR spectroscopy resolves resonances in 13
C
1
NMR spectra better than for H NMR spectroscopy, enabling
analysis of product ratios that would not be possible otherwise.
For analysis of small molecules, this technique can be employed
using little material (typically ≤10 mg), providing reliable
product ratios in <2 min using an instrument that operates at
Received: December 31, 2013
Published: March 6, 2014
© 2014 American Chemical Society
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Organic Letters
Letter
experiments that had short delays (2 s between pulses).18 As
long as equivalent resonances were compared, long relaxation
delays were not required to obtain satisfactory product ratios. It
was also not necessary to use inverse-gated decoupling: broad-
band decoupled spectra with short delays gave nearly identical
product ratios in approximately 1/15th of the time with better
signal-to-noise.19 A comparison between different methods is
shown in Table 1.
Table 2. Analysis of Camphor and Fenchone Mixtures
Highlighting the Acquisition Time Required To Obtain 13C
NMR Integration Data
a
experiment
experiment duration (min) mol % major component
1H (1 scan)
0.10
13
60.8
64.9
60.7
60.3
59.7
1H (128 scan)
13C (16 scan)
13C (512 scan)
13C (8192 scan)
1
Table 1. Diastereomeric Analysis of 2 by H and 13C NMR
1.8
29
Spectroscopy
a
experiment
1H (1 scan)
mol % major diastereomer
56.7
456
a
1H and 13C NMR spectra were acquired with a 2 s D1 on a total
1H (16 scan)
56.5
56.5
56.2
55.5
55.4
sample of 10 mg. 13C NMR spectra were acquired using an IGD pulse
program.
13C BBD (64 scan, 2 s D1)
13C BBD (64 scan, 30 s D1)
13C IGD (64 scan, 2 s D1)
13C IGD (64 scan, 30 s D1)
a
BBD = broadband decoupled, IGD = inverse-gated decoupled.
To integrate the spectra, we adhered to two important
constraints: (1) integrations of carbon resonances of two
different compounds would only be compared if the same
number of hydrogen atoms were present (to minimize
differences in NOE); (2) the ratios of integrals would be
averaged over all the different carbon atoms (this constraint is
20
1
similar to how one would integrate a H NMR spectrum).
All spectra described in the contribution were deconvoluted
to improve the quality of the integration. Deconvolution is a
commonly used technique to decrease the subjectivity and
increase the reliability of NMR spectral integrations.15,21 It is
rapid and easily implemented, effectively generating a spectrum
with a flattened baseline, which minimizes subjectivity during
integration.10 Deconvolution is not necessary to obtain accurate
13C NMR integrations, but the ease of its application
(automated on most instruments) is such that little additional
expertise on the part of the experimenter is required.
1
Figure 1. Mixtures of fenchone and camphor analyzed by H and 13
NMR spectroscopy and GC for comparison.
C
(Tables 3 and 4). This technique was accurate to within <2.5%
for sets of aliphatic compounds and cis-/trans-isomers.
Aromatic compounds and those containing heteroatoms were
analyzed accurately to within 3.4% of 1H NMR spectra
integration (single pulse). Accuracy improved for both sets
with longer D1 values of 30 s, although more significantly with
molecules containing aromaticity or heteroatoms.
Quantitative 13C NMR spectroscopy employing short D1
values can also be used to analyze molecules with different
connectivity. Analysis of the constitutional isomers camphor
Quantitative 13C NMR analysis employing short relaxation
delays can also be used for compounds of interest to materials
scientists and chemical biologists (Table 4). Two regioisomers
of a paracyclophane, which is an intermediate in the synthesis
of conjugated polymers,22 were analyzed by 13C NMR
1
and fenchone was performed by H NMR spectroscopy, GC,
and 13C NMR spectroscopy on samples with different ratios of
the two terpenes. For one particular mixture, the speed and
1
1
accuracy of H and 13C NMR spectroscopy were compared
spectroscopy, H NMR spectroscopy, and HPLC (Figure S-2
(Table 2). Using a sample that contained 10 mg of total sample,
decoupled 13C NMR spectroscopy provided a ratio of products
in <2 min that was virtually identical to the result obtained by
single-pulse H NMR spectroscopy. There was no benefit to
longer acquisition times for either spectroscopic technique.
in the SI). Presumably due to the difference in extinction
coefficients of the two regioisomers,22,23 the ratio found by
1
HPLC differed the most from the value obtained by H NMR
spectroscopy. The diastereomeric ratio of allyl lactide,24 with
only one resolvable signal by 1H NMR spectroscopy, was
determined by 13C NMR spectroscopy with eight resolvable
signals (the deconvoluted 13C NMR spectrum is shown in the
Table of Contents graphic). The knowledge about lactide
stereochemistry is important to polymer chemists, because the
tacticity of the resulting polymer determines its thermal
properties and crystallinity.25 The ratio of cis- and trans-
rotamers of a model peptoid26 was analyzed to assess the utility
of 13C NMR integration in the field of peptoid chemistry,27
where conformation governs function. The calculated energy
difference between the two amide rotamers determined by 13C
1
These techniques afforded comparable results for nearly all
1
product ratios analyzed (Figure 1). Integration of H NMR
spectra acquired with multiple scans and short relaxation delays
gave the poorest results (Table 2 and Figure 1), likely because
of selective saturation of nuclei.2 The mixtures containing
fenchone in 1 and 99 mol % by weight yielded ratios of 100:0
and 0:100 rather than 99:1 and 1:99, respectively, by
integration of 13C NMR spectra because the concentration of
the minor component was below the detection limit of 13C
NMR spectroscopy.
Integration of 13C NMR spectra acquired using short
relaxation delays is applicable to a range of different structures
and H NMR spectroscopy are within 0.03 and 0.01 kcal/mol,
1
respectively, of the values reported in the literature.28 The
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Organic Letters
Letter
Table 3. Analysis of Mixtures of Molecules Comparing the
Table 5. Influence of O2 on the Accuracy of 13C NMR
Spectroscopy
1
Accuracy of H and 13C NMR Spectroscopy
a
experiment
mol % major
1H (1 scan) with addition O2
69.2
69.3
69.9
66.8
1H (1 scan) without addition of O2
b
13C (64 scan) with addition of O2
b
13C (64 scan) without addition of O2
a
b
10 mg of sample. BBD, 2 s D1.
from 2.5% to 0.7% when the sample was prepared from solvent
that was saturated with O2 prior to sample preparation.
13C NMR spectroscopic integration employing short
relaxation delays can also be used to determine degrees of
polymerization (DPs) in polymers.34 Polymers have been
analyzed by quantitative solution 13C NMR spectroscopy
with32 or without relaxation agents,15,35,36 in particular to
gain insight into block copolymer composition,37 but rarely to
determine DPs.11,15 The number average molecular weight is
more commonly determined by 1H NMR spectroscopy,38
although spectral congestion can pose limitations. Other
techniques to determine DP are matrix-assisted laser
desorption−ionization time-of-flight spectrometry (MALDI-
ToF)39 or size-exclusion chromatography (SEC).40 MALDI-
ToF requires ionization of the polymer, which may lead to
fragmentation.39 It also does not allow for sample recovery.
SEC is an indirect method and inherently inaccurate for
complex architectures such as brush copolymers and miktoarm
polymers.40
a
b
1
Single scan H NMR spectroscopy. 13C NMR spectroscopy using
IGD pulse program with 10 mg of sample, 512 scans, and a 2 s D1.
Values in parentheses employed a 30 s D1.
Table 4. Analysis of Paracyclophane Regioisomers, Allyl
Lactide, Peptoid Rotamers, and Sugar Anomers
Analysis of three model low molecular weight polymers
demonstrated that quantitative 13C NMR spectroscopy with
short delays could be used to analyze compounds of molecular
weights up to 4000 Da. The model compounds were chosen to
display end groups according to the polymer backbone signals
of interest (Scheme 2) to avoid NOE differences.35,41,42
Scheme 2. Structures of Polymers Analyzed in This Study
Quantitative analyses of poly(lactic acid) (PLA) samples with
up to 30 repeat units, poly(4-methyl-oxazoline) (P(Ox))
samples with up to 20 repeat units, and a poly(norbornenyl
octanoate) (P(NO)) with 17 repeat units were performed
successfully without the addition of relaxation agents. The data
are summarized in Table 6 and are in good agreement with 1H
NMR end group analysis and MALDI-ToF spectrometry. 13C
NMR spectra of P(Ox) also provide insight into the
microstructure of the polymer in solution: distinct signals are
observed for both cis- and trans-rotamers of the terminal
monomer subunit.
a
b
c
10 mg of sample in total. Single scan. BBD, 512 scans, and 2 s D1.
d
e
BBD, 1024 scans, and 2 s D1. BBD, 512 scans, and 2 s D1. Values in
parentheses employed a 30 s D1.
anomeric ratio29 of carbohydrates can also be quantified by 13
NMR spectroscopy.
C
To address the lower accuracy observed with some molecules
containing aromatic carbons (3.4%), we added a paramagnetic
relaxation agent.11,13 For these studies, we employed a mixture
of 3,5- and 2,5-dimethylphenol (Table 5). Relaxation agents
reduce longitudinal relaxation times, therefore improving
quantitative 13C NMR spectroscopic analysis.30−32 Oxygen
(O2)33 was chosen to avoid sample contamination. Accuracy
compared to 1H NMR spectroscopic integration improved
In summary, integration of 13C NMR spectra acquired with
short relaxation delays is a convenient method for determi-
nation of compound ratios, and it complements other
commonly used techniques, particularly in cases when these
techniques are insufficient. 13C NMR integration employing
1
short D1 values affords a greater spectral dispersion than H
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Organic Letters
Letter
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Table 6. Analysis of Degree of Polymerization (DP)
Comparing the Accuracy of MALDI-ToF, 1H NMR, and 13
C
a
NMR Spectroscopy
a
polymer
MALDI-ToF
1H
13C
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PLA 1
27.9
31.8
14.7
18.4
17.9
27.0
30.9
9.8
27.3
31.2
10.9
17.7
18.6
PLA 2
P(Ox) 1
P(Ox) 2
P(NO)
15.3
19.6
a
BBD, 2 s D1.
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(19) The experiments reported in this paper use a 90° pulse in the
carbon channel. Attempts to modify this pulse width to improve
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accuracy due to the increase in signal-to-noise for molecules with
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NMR spectroscopy and is quantitative as long as differences in
NOEs and relaxation times are considered. Using standard
pulse sequences, including broadband decoupling, 13C NMR
spectroscopic techniques can be used to obtain accurate
integration ratios in <0.5 h on as little as 100 μmol of sample.
For both polymers and small molecules, the only limiting factor
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, spectra, and compound character-
ization. This material is available free of charge via the Internet
at http://pubs.acs.org.
́
(25) Martin, O.; Averous, L. Polymer 2001, 42, 6209.
(26) Nishiwaki, K.; Ogawa, T.; Shigeta, K.; Takahashi, K.; Matsuo, K.
Tetrahedron 2006, 62, 7034.
AUTHOR INFORMATION
Corresponding Authors
■
(27) Yoo, B.; Kirshenbaum, K. Curr. Opin. Chem. Biol. 2008, 12, 714.
(28) Gorske, B. C.; Stringer, J. R.; Bastian, B. L.; Fowler, S. A.;
Blackwell, H. E. J. Am. Chem. Soc. 2009, 131, 16555.
(29) Dondoni, A.; Marra, A. Chem. Rev. 2000, 100, 4395.
(30) Kitagawa, T.; Okazaki, T.; Komatsu, K.; Takeuchi, K. J. Org.
Chem. 1993, 58, 7891.
(31) Okazaki, T.; Terakawa, E.; Kitagawa, T.; Takeuchi, K. J. Org.
Chem. 2000, 65, 1680.
(32) Rego, R.; Adriaensens, P. J.; Carleer, R. A.; Gelan, J. M. Polymer
*E-mail: marcus.weck@nyu.edu.
*E-mail: kwoerpel@nyu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health, National Institute of General Medical Sciences (GM-
61066). The Bruker Avance-400 MHz NMR spectrometer was
acquired through support of the National Science Foundation
(CHE-01162222). The MALDI-ToF MS was acquired through
the National Science Foundation (CHE-0958457). D.E.B.
acknowledges the Margaret and Herman Sokol Fellowship.
The authors thank Anderson J. Bonon, Dr. Elizabeth Elacqua,
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(33) Prosser, R. S.; Evanics, F. In Modern Magnetic Resonance; Webb,
G., Ed.; Springer: Netherlands, 2006; p 475.
(34) Although the systems exhibit MW values of 1000−4000 Da,
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to 30 are consistent with polymers; thus, the method can be applied
facilely to oligomers and low molecular weight polymers.
(35) Zhou, Z.; Kummerle, R.; Qiu, X.; Redwine, D.; Cong, R.; Taha,
̈
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Dr. Angie Garcia, Jie Lu, and Dr. Elizabeth Valentın (NYU) for
assistance.
A.; Baugh, D.; Winniford, B. J. Magn. Reson. 2007, 187, 225.
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