Anal. Chem. 1999, 71, 2301-2306
Co n s is t e n c y o f NMR a n d Ma s s S p e c t ro m e t ry
De t e rm in a t io n s o f Na t u ra l-Ab u n d a n c e S it e -S p e c ific
Ca rb o n Is o t o p e Ra t io s . Th e Ca s e o f Glyc e ro l
Be n-Li Zha ng,*,† Mic he l Trie rw e ile r,† Ca the rine J ouitte a u,† a nd Ge´ ra rd J . Ma rtin‡
LAIEM, UPRES-A Q6006, Universite´ de Nantes-CNRS, 2 rue de la Houssinie`re, BP 92208, 44322 Nantes Cedex 03,
France, and CEAIS, Site de la Ge´raudie`re, Rue P. A. Bobierre, BP 72304, 44323 Nantes Cedex 3, France
of sites i are often accessible with levels of precision compatible
with the natural dispersion. Although carbon-13 is about 70 times
more abundant than deuterium, the NMR determination of carbon
isotope ratios is less favorable due, in particular, to relatively large
relaxation times and to perturbations introduced by imperfect
broad-band decoupling, possibly associated with incomplete sup-
pression of nuclear Overhauser effects. Taking into account the
relatively small range of variation of the carbon isotope ratios at
natural abundance (currently less than 40‰ as compared to more
Quantitative determinations of natural-abundance carbon
isotope ratios by nuclear magnetic resonance (SNIF-NMR)
have been optimized by appropriate selection of the
experimental conditions and by signal analysis based on
a dedicated algorithm. To check the consistency of the
isotopic values obtained by NMR and mass spectrometry
(IRMS) the same glycerol samples have been investigated
by both techniques. To have access to site-specific isotope
ratios by IRMS, the products have been degraded and
transformed into two derivatives, one of which contains
carbons 1 and 3 and the other carbon 2 of glycerol. The
sensitivity of the isotopic parameters determined by IRMS
to fractionation effects possibly occurring in the course
of the chemical transformations has been investigated,
and the repeatability and reproducibility of both analytical
chains have been estimated. The good agreement ob-
served between the two series of isotopic results supports
the reliability of the two different approaches. SNIF-NMR
is therefore a very attractive tool for routine determination,
in a single nondestructive experiment, of the carbon
isotope distribution in glycerol, and the method can be
applied to other compounds. Using this method, the
isotopic distributions have been compared for glycerol
samples, obtained from plant or animal oils, extracted
from fermented media, or prepared by chemical synthe-
sis. Typical behaviors are characterized.
2
than 500‰ for H), very high levels of experimental accuracy are
required. It was shown in 1991 that reliable values could be
obtained in favorable cases,4 but significant progress is expected
from improvements in the technical performance of the spec-
trometers and from the development of dedicated algorithms for
signal analysis.5
Isotope ratio mass spectrometry (IRMS) is recognized as a
sensitive and precise method of determination of natural-
abundance carbon isotope ratios. However, since the product is
combusted prior to the determination, only an overall molecular
value is measured on the resulting CO2. In practice, site-specific
natural-isotope ratios, (13C/ 12C)i, are accessible by IRMS but
suitable degradation reactions of the product are required in order
to isolate the different molecular positions.6-8 These transforma-
tions must be carried out under conditions devoid of spurious
isotope effects or under conditions where appropriate corrections
can be properly estimated. This IRMS strategy has been used in
the case of glycerol, and large deviations with respect to a
statistical distribution of carbon-13 among the three carbon sites
have been measured.9 A recent SNIF-NMR investigation of (13C/
12C)i ratios of glycerol samples from various origins has deter-
mined trends in the isotopic distribution that are in good
agreement with the independent IRMS observations.10 Conse-
quently, it may be expected that the NMR technique has reached
The investigation of site-specific natural-isotope fractionation
by nuclear magnetic resonance (SNIF-NMR)1 has now been
widely exploited in different areas such as the determination of
kinetic or thermodynamic isotope effects,2 the study of biochemi-
cal pathways,3 and the origin inference of various materials.2 In
this respect, deuterium NMR was shown to be well suited to the
quantification of monodeuterated isotopomers at natural abun-
dance, and the isotope ratios, (D/ H)i, of molecular sites or clusters
(4) Caer, V.; Trierweiler, M.; Martin, G. J.; Martin, M. L. Anal. Chem. 1 9 9 1 ,
63, 2306-2313.
(5) Martin, Y. L. J. Magn. Reson., Ser. A 1 9 9 4 , 111, 1-10.
* To whom correspondence should be addressed. E-mail: benli.Zhang@
chimbio.univ-nantes.fr.
(6) Abelson, P. H.; Hoering, T. C. Proc. Natl. Acad. Sci. U.S.A. 1 9 6 1 , 47, 623-
632.
† LAIEM.
(7) De Niro, M. J.; Epstein, S. Science 1 9 7 7 , 197, 261-263.
(8) Monson, K. D.; Hayes, J. M. Geochim. Cosmochim. Acta 1 9 8 2 , 46, 139-
‡ CEAIS.
(1) Martin, G. J.; Martin, M. L. Tetrahedron Lett. 1 9 8 1 , 2, 3525-3528.
(2) Martin, G. J.; Martin, M. L. In NMR Basic Principles and Progress; Gu¨ nther,
H., Ed.; Springer-Verlag: Heidelberg, Germany, 1990; Vol. 23, pp 1-61.
(3) Martin, G. J.; Zhang, B. L.; Naulet, N.; Martin, M. L. J. Am. Chem. Soc.
1 9 8 6 , 108, 5116-5122.
149.
(9) Weber, D.; Kexel, H.; Schmidt, H. L. J. Agric. Food Chem. 1 9 9 7 , 45, 2042-
2046.
(10) Zhang, B. L.; Buddrus, S.; Trierweiler, M.; Martin, G. J. J. Agric. Food Chem.
1 9 9 8 , 46, 1374-1380.
10.1021/ac9812375 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/07/1999
Analytical Chemistry, Vol. 71, No. 13, July 1, 1999 2301