NMR SPECTROSCOPY APPLICATIONS / Food 303
Table 19 Ranges of some spin spin coupling constants for Cd
Further Reading
and Hg organometallic compounds
J Complex type/ligands Range (Hz) Gielen M, Willem R, and Wrackmeyer B (eds.) (1997)
Advanced Applications of NMR to organometallic
2
J(113Cd,1H) Alkyls 49 90
Chemistry, Physical Organometallic Chemistry, vol. 1.
1
J(113Cd,13C) Alkyls, CN 271 1060
3 New York: Wiley.
J(113Cd,1H) Alkyls 6 70
n Kidd RG (1971) NMR spectroscopy of organometallic
J(113Cd,13C) Alkyls 17 45
2
chemistry. In: Tsutsui (ed.) Characterization of Organo-
J(199Hg,1H) Alkyls 94 265
3
metallic Compounds, part II, ch. 8. New York: Wiley-
J(199Hg,1H) Organomercurials 38 311
1
J(199Hg,13C) Alkyls, aryls, CN 264 3875 Interscience.
2
13
J(199Hg,13C) Alkyls, aryls 24 920
Mann BE (1974) C NMR chemical shifts and coupling
3
J(199Hg,13C) Alkyls, aryls 93 205
constant of organometallic compounds. Advances in
2
J(199Hg,19F) Vinyls, alkyls 338 1911
Organometallic Chemistry 12: 135 213.
1
J(199Hg,31P) PR3 1980 12 970 13
Mann BE (1974) C NMR spectroscopy of organo-
transition metal complexes. In: Axenrod T and Webb
GA (eds.) NMR spectroscopy of Nuclei other than pro-
intermediate mercury acceptors, and so far the latter
tons, ch. 11: 153 156. New York: Wiley-Interscience.
does not correspond to the behavior of d(199Hg). In
2
Mann BE (1988) Recent developments in NMR spectro-
bis-fluoroalkyl mercury derivatives J(199Hg,19F) de-
scopy of organometallic compounds. Advances in Organo-
creases as the donor ability of the solvent increases.
metallic Chemistry 28: 397 457.
2 3
In CdMe2, J(113Cd1H) is positive, but J(113Cd1H)
Mann BE, et al. (1985) Journal of the Chemicl Society,
2
has the opposite sign. Although J(199Hg1H) is
Dalton Transactions: 693.
negative in alkyl derivatives of mercury it is positive
Orrell K (1999) Dynamic NMR spectroscopy in inorganic
19
in vinyl derivatives. Couplings to F in Hg CF3 and organometallic chemistry. Annual Reports on NMR
1
compounds have the opposite sign to those to Hin
Spectroscopy 37: 1 74.
the analogous Hg Me system.
Roc (1987) CD spectroscopic characterization of inorganic
and organometallic complexes by metal and high-pres-
sure NMR. In: Wayda AL and Darensbourg MV (eds.)
See also: Nuclear Magnetic Resonance Spectroscopy:
Experimental Organometallic Chemistry, ACS Symposi-
Overview; Principles. Nuclear Magnetic Resonance
um Series No. 357, ch. 8. Washington, DC: American
Spectroscopy-Applicable Elements: Hydrogen Iso-
Chemical Society.
topes; Carbon-13; Fluorine-19; Nitrogen-15; Phosphorus-
Vrieze K and Van Leeuwen PWNM (1971) Studies of dyna-
31. Nuclear Magnetic Resonance Spectroscopy Ap-
mic organometallic compounds of the transition metals
plications: Proton NMR in Biological Objects Subjected
by means of NMR. Progress in Inorganic Chemistry 14:
to Magic Angle Spinning. Nuclear Magnetic Resonance
1 63. New York: Wiley Interscience.
Spectroscopy Techniques: Solid-State; Surface Coil.
NUCLEAR MAGNETIC RESONANCE
SPECTROSCOPY APPLICATIONS
Contents
Food
Forensic
Pharmaceutical
Proton NMR in Biological Objects Subjected to Magic Angle Spinning
Introduction
Food
Food materials exhibit a wide range of states of
complexity. These range from homogeneous and re-
C Deleanu, National NMR Laboratory and Institute of
latively simple mixtures, as in edible oils, to quite
Organic Chemistry, Bucharest, Romania
complex heterogeneous systems, as in meat or bread
& 2005, Elsevier Ltd. All Rights Reserved. (in the latter case both air and heterogeneous solid
304 NMR SPECTROSCOPY APPLICATIONS / Food
phases are present). This range of physical states the determination of the structure of various com-
and chemical compositions requires a wide palette of pounds isolated from all types of foodstuff. This ap-
nuclear magnetic resonance (NMR) techniques and plication is an essential tool in advanced food
approaches for obtaining in-depth information and research and requires prior isolation and purifica-
characterization of various foodstuffs. NMR tech- tion of the compound under study.
niques are versatile and can provide different types As this approach is identical to that employed in
of data and information on the same sample, depen- synthetic chemistry, we would not cover it in this
ding on the experimental parameters selected. The article. The interested reader will find extensive
applications include qualitative and quantitative coverage of NMR techniques for structure determi-
analysis monitoring of processes or biochemical re- nation in other articles of this encyclopedia.
actions determination of the structure of isolated The following sections will provide examples of
compounds, the mobility and state of fat and water, applications of various NMR techniques to mixtures
the microstructure and aggregation state of various of compounds in various food matrices or fluids.
components in foodstuffs, as well as macroscopic
imaging with all the advantages of obtaining cross
Fruits and Fruit Juices
sections without the need for cutting the sample. In
1
terms of quantifying various compounds, NMR H-NMR spectra for various fruit juices have been
spectroscopy has the ability to provide the global published, and signals have been assigned to sugars,
concentration of the sample and not only the surface amino acids, and other compounds like alcohols, ac-
1
concentration. The disadvantages of the early days ids, and polyphenols. Furthermore, H-NMR spectra
of NMR spectroscopy such as  low sensitivity or have been used in combination with chemometric
 expensiveness of the instrumentation do not hold techniques for discriminating between apple varieties
true in the early twenty-first century. Thus, the mod- and for detecting adulteration of orange and apple
ern hardware, software, automation, and hyphena- juices. There are several non-NMR methods classi-
tion have reduced the experimental time to a few cally employed for determination of fruit juice adul-
minutes even for two-dimensional (2D) experiments teration (density, total amino acids, flavonoid and
and increased the sensitivity, allowing easy detection
carotenoid composition). Some of these methods
of compounds at levels of micrograms and even (e.g., liquid chromatography (LC)) require however
nanograms. Moreover, there are arguments sup- standards of the suspected markers that are not
porting the fact that sometimes well established always available. Moreover, with the classical meth-
techniques like gas chromatography (GC) mass ods one should preselect the most likely markers for
spectrometry (MS) produce false results, several of
a particular sample based on an educated guess on
the  new compounds reported based on these tech- the expected type of adulteration. HR NMR has the
niques being in fact experimental artifacts resulting advantage of offering a global profile. Thus, even
from the decomposition of some original labile com- without a total assignment of signals, in conjunction
pounds in foodstuffs. In such cases NMR has the
with chemometric techniques the method can dis-
great advantage of providing information on the criminate between various patterns.
1
studied compounds under ambient conditions of Figure 1 shows examples of H-NMR spectra for
temperature and pressure. some common fresh fruit juices.
1
The typical profile of the H-NMR spectrum of
fruits and fruit juices could be rationalized by con-
High Resolution (HR) NMR (Frequency
sidering three regions. Between 0.5 and 3 ppm there
Domain NMR) are various signals generated by amino acids, other
carboxylic acids, and alcohols. Between 3 and
Most HR NMR food sciences analyses are done in
5.5 ppm the NMR spectrum is dominated by sugars
solution. However, the information provided by HR
as well as by the residual water signal (4.8 ppm).
solid-state NMR is also very valuable for food sci-
Although other compounds give rise to some signals
ences. Thus it is very likely that in the near future we
in this region, in most cases these signals are of little
will see a more balanced number of liquid- and solid-
practical use as they are overlapped by the sugar
state NMR studies in food sciences.
signals. The region between 5.5 and 10 ppm exhibits
signals generated by aromatic and heterocyclic com-
Elucidating the Structure of Isolated Compounds
pounds, formic acid, and aldehydes.
13
HR NMR is currently the most powerful technique Solid-state C-NMR has also been used to study
for elucidating the structure of isolated compounds fruits, for instance to follow changes in the cell wall
in solution. There are numerous papers describing polymers during ripening.
NMR SPECTROSCOPY APPLICATIONS / Food 305
7.5 7.0 6.5 ppm
(E) 876 54321 ppm
7.5 7.0 6.5 ppm
(D) 876 54321 ppm
7.5 7.0 6.5 ppm
(C) 876 54321 ppm
7.5 7.0 6.5 ppm
(B) 876 54321 ppm
7.5 7.0 6.5 ppm
(A) 876 54321 ppm
1
Figure 1 H-NMR spectra (400 MHz) for some common fresh fruit juices. (A) lemon juice; (B) grapefruit juice; (C) orange juice;
(D) apple juice; and (E) apricot pure.
306 NMR SPECTROSCOPY APPLICATIONS / Food
Vegetables
hypocholesterolemic, and hypoglycemic effects.
b-Glucan is isolated from yeast, and the established
1
The assignment of many components in the H-NMR
1
wet analytical methods are quite tedious. H-NMR
spectrum of tomato juice has been published recently.
has been proved to be a rapid alternative method.
1
The H-NMR profile of tomato juice resembles that
Sugars have been determined using both liquid-
of fruit juices, with the same distinct zones, i.e.,
and solid-state NMR in fruits, vegetables, and their
aminoacids, sacharides, and aromatic compounds.
1 juices, as well as in honey.
H-NMR spectra of methanolic extracts of control
Starch products and glycogen have been studied
and genetically modified varietals of tomatoes have
1 13 31
extensively using H-, C-, and P-NMR spectros-
been analyzed using chemometric techniques. It was
copy. NMR spectroscopy has been used for determi-
possible to assess variations in several metabolites.
ning the degree of polymerization, the average
1 2 13
H, H, and C solid-state magic angle spinning
number of glucose units, the branching degree, and
(MAS) NMR spectra have been recorded for tomato
the anomer distribution of the reducing sugars. The
skin and potato tissue, providing information on the
interaction of starch with water and the mobility of
structure and dynamics of the cuticle polyesters.
polysaccharide chains have been investigated
It has been demonstrated that for the case of some
through one-dimensional (1D) and 2D solid-state
flavor compounds present in garlic, NMR is superior
NMR experiments. The mobility of  freezable and
to GC MS as the latter produces false results due to
1
 unfreezable water in starch was studied using H
the decomposition of some labile compounds.
2
and H solid-state NMR. The aging (retrogradation)
Large differences in the ratio between proteins
of starch has been studied using cross polarization
and polysaccharides in various mushroom strains
13
magic angle spinning (CPMAS) C-NMR.
13
have been observed using C solid-state MAS NMR
1
Figure 2 presents the H-NMR spectrum of
spectroscopy.
glucose. As always, in solution glucose is formed by
a mixture of a- and b-anomers. The spectrum em-
Wine
phasizes the region between 3.2 and 4.0 ppm, which
Surprisingly, in contrast with the early success of
is very crowded in the case of saccharides, glucose
2
H-NMR spectroscopy in wine analysis (see Wine
being only one of the saccharides present in various
1
section of SNIF-NMR), H-NMR has not been used
foodstuffs. Thus, many food products (including
on whole wine samples until very recently. Although
fruits, vegetables, honey, and wines) exhibit an NMR
the possibility of quantifying methanol in wine was
spectrum crowded with signals in the saccharide
proved in the early 1990s, it was only a decade later
region.
that the field took off, with many minor compounds
identified in whole wine samples and with chemo-
Tea
metrics helping to rationalize the global profile of the
1
Several flavonoids isolated from tea have been analy-
H-NMR spectrum of wine samples. The same trend
zed and their structure determined using NMR.
is paralleled with spectra for other alcoholic beve-
There are several problems with the classical meth-
rages like beer.
ods of analysis of flavonoids in tea. Due to the pres-
Recently an interesting application used a home-
ence of complex mixtures of flavonoids in tea, they
made spectrometer based on a 310 mm bore magne-
are often characterized as  total polyphenols . The
tic resonance imaging (MRI) instrument operating at
1 13
colorimetric method for analysis of total phenols can
2 T to record H- and C-NMR spectra of intact
interfere with other reducing compounds. LC can
unopened wine bottles. Thus, NMR was able to
well resolve peaks for individual flavonoids; how-
detect spoiled bottles with acetic acid developed in
ever, there are only a few standards available com-
them without opening them. The application could
mercially, making the assignment of peaks uncertain
be very valuable for old and expensive wines.
in many cases. Thus, the structure of flavonoids
Sugars (Carbohydrates) giving rise to peaks in LC is often determined using
various 1D- and 2D-NMR experiments.
1 13
There are many studies involving H- and C-NMR
spectroscopy of carrageenans, a family of poly-
Coffee
saccharides with the structure of linear sulfated
galactans. They are extracted from certain species The early NMR studies of coffee were limited to
of red seaweed and have been used for a long time as elucidating the structure of isolated compounds.
natural texturing ingredients in the food industry. Several of the major constituents of espresso coffee
1
The polysaccharide b-glucan has been proved to have been identified in the H-NMR spectrum.
1
induce beneficial effects such as immunobiological, H-NMR data in combination with chemometrics
NMR SPECTROSCOPY APPLICATIONS / Food 307
5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 ppm
1
Figure 2 The H-NMR spectrum of a mixture of a- and b-glucose in D2O recorded at 400 MHz. The high peak at 4.8 ppm is the
residual water signal.
have been recently used for verifying the authenticity high resolution magic angle spinning (HRMAS)
of instant coffees from various producers. NMR spectroscopy.
Fats and Oils
Milk and Dairy Products
Edible fats and oils are ideal candidates for HR
The water methanol extracts of cheese have been
1
NMR. Their good solubility in organic solvents
analyzed using H-NMR, and assignments of several
makes the NMR analysis fast, easy, and accurate.
amino acids were done using 2D spectra. Variations
Issues such as the composition of fatty acids in oils
in the content of amino acids were observed as a
function of ripening time and distance from the cen- and fats, adulteration with cheaper oils (especially in
the case of extra virgin olive oil), degradation of oils
ter of the cheese wheel. The fatty acid triglycerides
1
and fats in time and at elevated temperatures, and
from milk have been analyzed using both H- and
13
content of fatty acids in single intact seeds (for
C-NMR.
31
breeding purposes) have been studied using NMR.
Liquid state P-NMR has been employed since a
long time back for studying milk and casein. Solid- NMR method has some advantages over other meth-
31
ods. Thus, there is no need for sample manipulation
state P-NMR has been used for studying the nature
before the analysis, information on the distribution
of micelles and the interaction between compounds
of the fatty acid chains in the glycerol moiety (po-
that form them.
sition 1/3 versus 2) is available, and the analysis has a
13
relatively high speed (even for C-NMR, results
Cereals and Bread
being obtained within minutes).
13 31 1
Solution-state C-NMR and P-NMR have been A typical H-NMR spectrum for sunflower oil is
employed in the study of lipids from corn suspension presented in Figure 3.
13
cells. Corn cells have also been grown in the presence A typical C-NMR spectrum for the same
13
of C-enriched acetate for following its incorpora- sunflower oil is presented in Figure 4.
tion into oleates and linoleates. Figure 5 presents details of the region 172
13 13
Solid-state CPMAS C-NMR has been used for 174 ppm in the C-NMR spectrum (100 MHz),
investigating changes in maize and sorghum proteins illustrating the power of the method in assigning
on wet cooking and popping. the distribution of fatty acids in the positions 1/3 and
The hydration of proteins from wheat seeds has 2 of the glycerol moiety, based on the signals of the
13 1
been studied using solid-state C CPMAS and H CO groups.
308 NMR SPECTROSCOPY APPLICATIONS / Food
ppm
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
1
Figure 3 H-NMR spectrum (400 MHz, CDCl3) of a sample of sunflower oil.
ppm
160 140 120 100 80 60 40 20
13
Figure 4 C-NMR spectrum (100 MHz, CDCl3) of a sample of sunflower oil.
If for most food applications, recording the NMR NMR spectra of edible oils and fats. The concentra-
spectra at the highest available magnetic field tion dependence on chemical shifts of model triglyce-
strength is a great advantage, for edible oils and fats rides has also been explored. Several papers deal with
there is not much improvement on passing from 300 authentic edible oil samples, with reference to both
to 600 MHz. In this case, the same types of groups assignment of NMR signals and authentication is-
from different long chain fatty acids produce similar sues. As expected, many papers deal with NMR of
1
signals in the H-NMR spectrum. This fact makes olive oils due to the high market value of the virgin
the routine medium-field (300 400 MHz) NMR and extra virgin qualities. A lot of effort is put in
spectrometers suitable and very competitive (in terms authentication and determination of the origin of
of price-to-quality ratio) for research and quality olive oils in connection with the Denomination of
control of edible oils. Protected Origin. Papers also deal with the identifi-
Individual fatty acids and esters have been used as cation of mono- and diglycerides in edible oils, as
model compounds for assignment of signals in the well as other compounds like phenolic derivatives.
NMR SPECTROSCOPY APPLICATIONS / Food 309
CO1/3
Linoleic
acid
CO2
Oleic
Linoleic
acid
acid
Saturated
Oleic
acids
acid
ppm
173.1 173.0 172.9 172.8 172.7
13
Figure 5 Enlargement of the carbonyl region in the C-NMR spectrum (100 MHz, CDCl3) of a sample of sunflower oil.
Polyunsaturated fatty acids (PUFAs), characteristic of temperatures. PUFAs are markers for the fish species,
fish lipids but also present in small quantities in age, freshness, storage, and processing conditions.
vegetable oils, have been also investigated using
Meat
NMR. Apart from the composition in fatty acids or
1
minor compounds, the CH region in H-NMR, and 13
C solid-state HRMAS NMR studies have been car-
13
the CH2 region in C-NMR have been used for de-
ried out on cartilages.
termining the iodine value. Several studies deal with 31
P-NMR spectroscopy studies of energy-related
issues like the changes induced in the composition of
metabolites have been carried both in HR NMR
oils by oxidation, heating, or hydrogenation.
instruments as biopsies and in MRI instruments as
entire muscle pieces. The phosphorus metabolites are
Seeds
directly linked to meat quality. The influence of
13
the stunning method on the postmortem energy me-
C-NMR spectra of intact seeds allow the identifi-
tabolism and meat quality has been explored
cation and quantitation of the main fatty acids.
31
13
using P-NMR. The pH has been determined using
Solid-state C HRMAS and distortionless en-
31
P-NMR and associated with the meat quality.
hancement by polarization transfer (DEPT) MAS
Although the pH is measured routinely through
NMR also have been used for studying integral
voltammetry, the NMR method has the advantage of
seeds. Thus, the three major components, fats, carbo-
measuring the pH in an inner part of the sample,
hydrates, and proteins (including the position of
being able to assess it both intra- and extracellulary,
the fatty acid in glycerol) could be identified and
as well as in vivo.
quantified.
31
In vivo P-NMR has been used for studying the
Eggs
metabolites during the ripening and drying of seeds.
The oxidation of cholesterol in egg powder has been
1 31
Fish
detected using H-NMR. P-NMR spectroscopy has
1 been used to quantitate phospholipids in egg lecithin.
Fish oils have been extensively investigated using H-
13
and C-NMR. PUFAs, important in diet, are be-
Essential Oils
lieved to reduce the risk of cardiovascular diseases.
NMR analysis of PUFA is an interesting alternative Essential oils are important flavors and aromas.
to the widely used GC and GC MS techniques when Many individual compounds have been isolated and
artifacts may arise either during transesterification characterized. Unseparated mixtures forming essen-
13
or within the GC MS equipment due to elevated tial oils have also been analyzed using C-NMR.
310 NMR SPECTROSCOPY APPLICATIONS / Food
Agricultural Chemistry
positions of an organic molecule depends on factors
such as the plant metabolism (thus bearing infor-
It has been shown that even with medium-field NMR
mation on the type of sugar that by fermentation
1
spectrometers (300 MHz), quantitative H- and
produces the ethanol) and the isotopic composition
31
P-NMR can rival with chromatography as an
of the ground and rain water (thus bearing infor-
analytical method for agricultural chemicals. The
mation on the geographic position). The isotopic
advantage of the NMR is that it does not require a
2
ratio H/1H in water varies on Earth between 90
reference standard as is the case with chromato-
and 160 ppm, depending on the latitude, with the
graphy. The possibility of detecting organophospho-
highest values at the equator. It has been proven
31
rus insecticide residues in crops using P-NMR has
that deuterium in the CH3 site of the ethanol
been demonstrated. Soil samples have been analyzed
molecule comes from sugars, whereas deuterium
1 13 31
using H, C, and P solid-state and solution-state
in the CH2 site comes mainly from water. Thus, in
NMR experiments.
2
the end, the isotopic ratio H/1H in different sites
of the distilled ethanol bears complex information
Plant and Cell Metabolism and
related to the grape type, geographical origin, cli-
Other Biotransformation
mate, production year, added sugar, or added
The metabolism of plants has been followed ex-
water. In order to improve the power of the meth-
tensively using various NMR techniques, employing
od, the isotopic composition in the two sites is no
1 31 13
H, P, and C nuclei. All kinds of samples, cells,
longer expressed as a relative ratio but as an abso-
tissues, and extracts, both in vivo and in vitro, have
lute ratio, calibrated against a standard of known
been investigated. A special hardware design is used
isotopic composition. The currently official standard
for following biotransformations in vivo. Thus the
is N,N-tetramethylurea (TMU). Typically fields of
living material is suspended in a culture medium in
the NMR tube and air is bubbled through a capillary
tube to maintain life.
CH2
TMU
2 CH3
H-NMR (Site Specific Natural
Isotope Fractionation Using NMR
(SNIF-NMR))
2
H SNIF-NMR is a high-resolution technique. OH
However, the success of this technique in food sci-
ences, together with the large number of published
papers, justifies treating it under a separate section.
(A) 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm
Wine
2
Wine analysis using H-NMR is one of the major
OH
+
CH3
applications of NMR in food sciences, probably the
H2O CH2
most important application of HR NMR.
The basis for authentication of the geographic
origin of wine and for spotting its adulteration is
deuterium (2H) NMR spectroscopy. The method
(B) 7 6 5 4 3 2 1 ppm
developed in the early 1980s by Gerard J. Martin is
known as SNIF-NMR. The method was adopted by
the French government and later as an official meth-
od by the EU and by the Office International de la
CH3
CH2
Vigne et du Vin. SNIF-NMR was registered as a
trademark by Eurofins Scientific (Nantes, France).
The principle of the method consists in comparing
the ratios of the signals from the CH3 and CH2
2
groups of the ethanol molecule in the H-NMR (C) 75 70 65 60 55 50 45 40 35 30 25 20 ppm
spectrum. The method requires prior distillation of 2
Figure 6 HR NMR spectra of ethanol recorded at 9.4 T. (A) H-
1 13
the ethanol from wine. The power of the method
NMR (61 MHz); (B) H-NMR (400 MHz); and (C) C-NMR
2
relies on the fact that the H/1H ratio in various (100 MHz).
NMR SPECTROSCOPY APPLICATIONS / Food 311
400 or 500 MHz are used in routine analysis; natural versus synthetic origin of aromas, vari-
however, owing to the good separation of deute- ous essential oils, terpenoids, and others. There is a
rium signals, 300 MHz instruments can equally clear legislative discrimination between  chemically
provide reliable results. identical and  natural food aromas and flavors, the
2
Figure 6 presents the H-NMR spectrum of et-  natural ones being premium products. Owing to
hanol, employed in the SNIF-NMR method, and for the great difference in price between such natural and
1 13
comparison the H- and C-NMR spectra. synthetic aromas, the adulteration techniques be-
SNIF-NMR is also applied to a whole range of came more and more sophisticated. Thus, vanilla
alcoholic beverages, including: cognac, whisky, beer, adulteration techniques included even the addition
13 2
and grape must. of C- and H-enriched vanillin. Consequently, the
analytical methods have had to be improved over
2
the years, and presently combinations of H SNIF-
Fruit Juices
13
NMR, C IRMS, and chemometrics applied to
SNIF-NMR is already a well established technique several marker compounds in the flavor can success-
for fruit juices, being suitable for tracing both their fully cope with the most sophisticated types of adul-
geographical origin and adulteration. The method teration used to date.
2
was originally based exclusively on H/1H analysis,
but later its discrimination power was greatly in-
13
creased by using it in combination with C/12C
Hyphenated Techniques
analysis through isotopic ratio mass spectrometry
(IRMS). In the case of fruit juices SNIF-NMR is Emerging hyphenated techniques have already
mostly used for detecting adulteration with cheaper proved their analytical value and in the near future
sugars, being accepted as an Association of Official will certainly become common techniques in food
Analytical Chemists (AOAC) method. sciences. Commercially available equipment include
LC-NMR in both on-flow and stop-flow versions,
LC NMR MS, LC NMR UV-MS, and more recent-
Miscellaneous Applications of SNIF-NMR
ly hyphenation with solid-phase extraction (LC solid
Apart form wine and juices the method has been suc- phase extraction NMR).
cessfully applied to issues such as the authentication Figure 7 illustrates the power of the on-flow liquid
of sugars of various origins, origin of glycerol (sugar chromatography (LC) NMR technique in the ana-
fermentation versus edible oil transesterification), lysis of grape juice.
RT (min)
RT 26 min
30
20 RT 16 min
RT 13 min
10
RT 6 min
0
8.0 7.5 7.0 6.5 6.0
(C) 8.0 7.5 7.0 6.5
ppm
(B) ppm
8.0 7.5 7.0 6.5 ppm
(A)
1
Figure 7 On-flow LC NMR spectrum of a sample of grape juice. (A) Detail of the aromatic region in the normal H-NMR spectrum;
(B) on-flow LC NMR spectrum; (C) rows at different retention times (RTs) correspond to NMR spectra of individual compounds in the
mixture. (Reproduced with permission from Bruker BioSpin, Rheinstetten, Germany.)
312 NMR SPECTROSCOPY APPLICATIONS / Food
The SFC is a critical parameter for the fats and oils
Low-Resolution (LR) NMR (Time
industry. The official American Oil Chemists Society
Domain NMR)
(AOCS) wet method is dilatometery. Alternative wet
LR-NMR is the most commonly used NMR tech-
methods are differential thermal analysis and differ-
nique to date for quality control in food science and
ential scanning calorimetry (DSC). LR NMR was
industry, with several official quality control methods
proved to be an alternative method for SFC determi-
in force. The success of the technique is due to
nation in late 1950s. The early continuous wave LR
several factors, including the power of the method (in
NMR spectrometers rapidly found their way into the
terms of information and speed), the early applica-
fats and oils industry, the method being accepted by
tion of the method to foodstuffs, the ecological ap-
the Instrumental Techniques Committee of the AOCS
peal (no longer needing polluting chemicals), and the
as early as in 1972. Presently the technical choice is
relatively low cost of the equipment, making it a very
radio frequency (RF) pulsed LR NMR. Pulse NMR
attractive alternative to the tedious wet chemical
spectrometers are more compact, very efficient, and
methods.
relatively cheap. They have the advantage of exciting
the protons in the whole sample at once.
Fats and Oils
The determination of SFC has been extended to
oil-in-water emulsions, where additional experimen-
Solid fat content (SFC) analysis is probably the most
tal parameters have to be considered in order to dis-
used LR-NMR application in the food industry. The
tinguish between oil and water protons. The droplet
initial success of the method prompted Unilever
size of oil-in-water emulsions is usually measured
(manufacturer of margarines and related products)
using optical diffraction techniques, but it has also
and Bruker (manufacturer of NMR instruments) to
been determined through diffusion NMR experi-
start a joint venture with the goal of building a table-
ments.
top LR-NMR spectrometer for solid-to-liquid ratio
The degradation of fats and oils during frying has
analysis in the fat industry. The method was
also been determined using LR NMR. During frying
developed in the early 1970s and over the years
oils undergo complex chemical reactions such as
leaded to various quality control protocols for fat
oxidation, polymerization, hydrolysis, and isomeri-
and oils that are by now adopted as official methods
zation. During these processes polar groups accumu-
by various international and national organizations.
late. The official International Union of Pure and
The success story of SFC analysis opened wide the
Applied Chemistry and AOAC method for assessing
door for LR NMR methods to penetrate as routine
the frying fat quality is the determination of total
techniques in the food industry.
polar groups through preparative column chro-
As the relaxation time depends on the mobility of a
matography. Based on the fact that the relaxation
particular system (with solids having the shortest re-
time is decreasing in direct relationship with the
laxation times), it is easy to quantify mixtures using
amount of polar groups present in the sample, it has
NMR. The bulk magnetization decay (free induction
been proved that LR NMR data correlate well with
decay (FID)) for samples containing various solid-to-
the values obtained using the official column chro-
liquid ratios is presented in Figure 8.
matography method. The crystallization kinetics of
fats is best studied using LR NMR, and as a result
most of the published studies follow the crystalliza-
tion using either LR NMR or DSC.
100% liquid + 0% solid
Seeds
70% liquid + 30% solid
The wet method for determining fats in seeds is using
extraction with organic solvents. LR NMR revolut-
ionized the analysis of seeds, allowing rapid and
30% liquid + 70% solid
nondestructive analysis without the need for weigh-
ing and drying them. Thus the seeds could be further
used for selection in order to improve the genetic
0% liquid + 100% solid
capacity of oil production.
Time The principle of separating and quantifying vari-
ous components in a system based on the T2 relax-
Figure 8 Bulk NMR signal amplitude for samples with various
ation time is the following. Various components have
solid-to-liquid ratios. The signal decays exponentially with a con-
stant T2 relaxation time. various relaxation times mainly due to differences in
Amplitude
NMR SPECTROSCOPY APPLICATIONS / Food 313
Proteins,
sugars,
moisture,
and
oil
Oil
Moisture
Water
and oil
Oil
(A)
(B) (C)
(B) Time
Time
Figure 9 Effect of different drying methods on FID from sun-
180°
90°
flower seeds. The final moisture content is different when drying
takes place at 1001C (A), 601C (B), or room temperature (C).
mobility (with solids having the shortest relaxation
times). For instance, in seeds proteins and carbohy-
drates relax fast, and they mainly contribute to the
first part of the NMR signal, the free induction decay
(A) Time
(FID). Water has an intermediate relaxation time,
Figure 10 The spin-echo RF pulse sequence (A) and its result
whereas oil has the longest relaxation time. Thus, by
on the amplitude of the NMR signal (B) when applied to corn
carefully selecting the region of the FID where the
samples.
component of interest is making the major contribu-
tion, one can quantify this component relative to
content in intact fruits. The technique is suitable for
other components. Meanwhile the intensity of the
following the ripening process.
signal is proportional to the number of nuclei (pro-
tons) generating that signal. Thus by comparing the
Sugars (Carbohydrates) and Starch
intensity of FID in the region of the component of
interest for different samples or in comparison with a
LR NMR is a routinely used technique for determi-
calibrated standard, one can obtain quantitative re-
nation of water content in sugars.
sults. Figure 9 illustrates how water in various seeds
Starch hydration, water mobility, and the effect of
can be quantified with LR NMR when analyzing the
chemical modification on molecular mobility of
middle part of the FID signal. Thus, by drying seeds
starch have also been studied using LR NMR.
at room temperature, the quantity of water that re-
Alteration of starch over time (retrogradation) has
mains is higher (and the NMR signal s amplitude is
been studied using LR NMR. Most of the studies
1
higher) than by drying the same seeds at 60 or 1001C.
employed the T2 relaxation curves of H-NMR.
An almost identical curve was obtained on drying
Thus it was shown that the number of protons in the
various seeds (peanut kernels, mustard, sunflower,
solid phase increases with aging, this being explained
and soybean) by sun and at 1051C.
by the recrystallization of starch.
When it is desired to quantify the slowly relaxing
oil component, a more suitable pulse sequence Milk and Dairy Products
scheme would be the spin-echo. As illustrated in
The state of water in milk, milk powder, casein, whey
Figure 10, the time at which the second refocusing
proteins, and cheese has been investigated using LR
pulse (of 1801) is applied can be selected so as to
NMR.
obtain an NMR signal generated exclusively by oil.
The oil content can be accurately quantified when the
Chocolate
amplitude of the signal is compared with a calibra-
LR techniques are routinely used for determination
tion curve generated by standard samples.
of the fat content and solid-to-liquid ratio in choco-
late and cocoa products. Analysis of the fluidifica-
Fruits
tion of cocoa butter using LR NMR enabled the
LR NMR has been used to determine the changes in identification of cocoas according to the process and
water mobility in dehydrated fruits, and the sugar the type of roaster used.
Amplitude
Amplitude
RF
314 NMR SPECTROSCOPY APPLICATIONS / Food
Meat
LR NMR has been widely used in meat studies
for quite a long time. There are several advantages
associated with NMR, for instance the fact that
NMR measures the whole volume of the sample,
being less affected by surface effects. The total
water and its state in meat (e.g., bounded versus
1
free) can be estimated using LR H-NMR. This dif-
ference is evident on cooling when the free water is
freezing, leading to a shortening of T1 (longitudinal
relaxation time). The water holding capacity is an
important parameter of meat that could be estima-
ted using NMR. LR NMR has also been correlated
with pH and cooking loss using chemometrics. LR
NMR is currently a standard method for estimating
the total fat content in meat. This way the meat
needs not to be dried before the NMR analysis.
Types of tissue in calf and cow have been differen-
tiated using T2 LR NMR of water based on different
Figure 11 MRI cross-sections through a kumquat fruit at 7 T.
types of collagen present in various types of tissue.
(Reproduced with permission from Bruker BioSpin, Rheinstetten,
Another important parameter of meat is the
Germany.)
development of flavor associated with a long storage
time and storage temperature. The so-called
warmed-over flavor (WOF) is produced by autoxi-
NMR Imaging (Spatial Domain NMR)
dation of membrane phospholipids and degradation
The current MRI techniques provide information not
of proteins and heteroatomic compounds. Some
only on the internal structure of various foodstuffs
WOFs have been predicted using LR NMR and
but also on the distribution of various compounds
chemometrics.
(chemical shift imaging), mobility and diffusion of
various compounds (like water, oil, or compounds
Bread from various packing materials), or dynamic changes
like crystallization.
LR NMR can monitor the distribution and mobility
The technique is increasingly being used in food
of water during bread making (dough, baking to
sciences but still less than its true potential. The ex-
bread, staling).
planation for the delay in widespread use of NMR
The baking process has been performed inside the
imaging techniques in food science and industry is
1
NMR magnet and followed using the H T2 relax-
the high cost of the equipment.
ation time. The water mobility during storage of
Figure 11 shows four MRI cross-sections through
bread has been studied using the T1 and T2 relaxa-
a kumquat fruit. They start from near the surface
tion curves. The texture parameters (elasticity and
and advance toward the center of the fruit. The fruit
firmness) have correlated well with T2 curves of
flesh in the centre is surrounded by the peel. The
bread crumbs.
tissue between the fruit flesh and the peel is not
visible because of a very short T2 relaxation time.
Spherical structures in the peel are visible, and they
Potatoes
contain the typical aromatic contributions of the
Prediction of the sensory texture properties of
fruit.
cooked potatoes, based on raw potato analysis, is
MRI has been applied to various foodstuffs such as
of major importance to the food industry. The sen-
meat, cereals, seeds, fruits, vegetables, cheese, or
sory texture quality of potatoes has been predicted
chocolate. The distribution and mobility of various
using T2 LR NMR. Correlation of the T2 LR NMR
constituents including water, sugars, and lipids have
data with the chemical composition of potatoes has
been monitored.
also been performed using chemometrics. Differen-
tiation between potato varieties and determination of
See also: Carbohydrates: Overview; Sugars  Spectro-
dry matter content has also been done using T1 and photometric Methods. Food and Nutritional Analysis:
Overview; Coffee, Cocoa, and Tea; Alcoholic Beverages;
T2 LR NMR.
NMR SPECTROSCOPY APPLICATIONS / Forensic 315
Meat and Meat Products; Dairy Products; Vegetables and
Analytical Methods of Food Authentication, pp. 36 75.
Legumes; Oils and Fats; Fruits and Fruit Products. Lip- London: Blackie Academic and Professional.
ids: Overview; Fatty Acids. Liquid Chromatography:
Deleanu C and Paré JRJ (1997) Nuclear magnetic reso-
Liquid Chromatography Nuclear Magnetic Resonance
nance. Principles and applications. In: Paré JRJ and
Spectrometry. Mass Spectrometry: Overview. Nuclear
Bélanger JMR (eds.) Instrumental Methods in Food
Magnetic Resonance Spectroscopy: Overview; Princi-
Sciences, pp. 179 237. Amsterdam: Elsevier.
1
ples; Instrumentation. Nuclear Magnetic Resonance
Guillen MD and Ruiz A (2001) High resolution H nuclear
Spectroscopy-Applicable Elements: Hydrogen Iso-
magnetic resonance in the study of edible oils and fats.
topes; Carbon-13. Nuclear Magnetic Resonance Spec-
Trends in Food Science & Technology 12: 328 338.
troscopy Techniques: Solid-State; In Vivo Spectroscopy
Guillou C, Remaud G, and Martin GJ (1991) Applica-
Using Localization Techniques.
tion of deuterium NMR and isotopic analysis to the
characterization of foods and beverages. Trends in Food
Science & Technology 2: 85 89.
Further Reading
McCarthy MJ (1994) Magnetic Resonance Imaging in
Foods. New York: Chapman & Hall.
Beauvallet C and Renou JP (1992) Applications of NMR
Rutledge DN (2001) Characterization of water in agro-
spectroscopy in meat research. Trends in Food Science &
food products by time domain-NMR. Food Control 12:
Technology 3: 241 246.
437 445.
Belloque J and Ramos M (1999) Application of NMR
1 13
Sacchi R, Addeo F, and Paolillo L (1997) H and CNMR
spectroscopy to milk and dairy products. Trends in Food
of virgin olive oil. An overview. Magetic Resonance in
Science & Technology 10: 313 320.
Chemistry 35: S133 S145.
Belton PS, Delgadillo I, Gil AM, and Webb GA (eds.)
Tiwari PN and Gambhir PN (1995) Seed oil determination
(1994) Magnetic Resonance in Food Science. Cambridge:
without weighing and drying the seeds by combined free
Royal Society of Chemistry.
induction decay and spin-echo nuclear magnetic reso-
Belton PS, Delgadillo I, Holmes E, et al. (1996) Use of
1
high-field H-NMR spectroscopy for the analysis of liq- nance signals. Journal of the American Oil Chemists
uid foods. Journal of Agricultural Food Chemistry 44: Society 72: 1017 1020.
1483 1487. Todt H, Burk W, Guthausen G, et al. (2001) Quality con-
Belton PS, Hills B, and Webb GA (eds.) (1999) Advances in trol with time-domain NMR. European Journal of Lipid
Magnetic Resonance in Food Sciences. Cambridge: The Science and Technology 103: 835 840.
Royal Society of Chemistry. Vlahov G (1999) Application of NMR to the study of olive
Colquhoun IJ and Lees M (1998) Nuclear magnetic reso- oils. Progress in Nuclear Magnetic Resonance Spectro-
nance spectroscopy. In: Ashurst PR and Dennis MJ (eds.) scopy 35: 341 357.
Forensic
B Dawson, Health Canada, Ottawa, ON, Canada
known to be a powerful tool for the elucidation of
chemical structures and the identification of organic
Canadian Crown Copyright & 2005. Published by Elsevier Ltd. All
compounds. It has been used in various types of
rights reserved.
forensic analysis for many years. It should be stressed
that NMR is not used as a stand-alone technique in
the forensic laboratory, but in conjunction with
Introduction
many others, including mass spectrometry, infrared
Since conclusions reached in forensic laboratories are (IR) spectroscopy, and various chromatographic
used in the criminal justice system, they must leave techniques.
no room for doubt. Thus, analytical methods must Some early reports on investigations using NMR
meet strict criteria. They must be extremely selective, spectroscopy for forensic analysis were pessimistic
reproducible, sufficiently sensitive, and suitable for about its application as a routine method. NMR was
qualitative and quantitative analysis. It is also highly viewed as an insensitive technique when compared
desirable for the method to call for the minimum with some of the other methods of analysis; the cost
number of pretreatment steps and to be applicable to of the instrument was comparatively high and the
compound mixtures without preliminary separation results obtained required considerable expertise to
of their components. Nuclear magnetic resonance interpret. This situation no longer exists. During the
(NMR) spectroscopy meets these criteria. It is well last several years, the amounts of material required