NMR in Food

intermediate mercury acceptors, and so far the latter
does not correspond to the behavior of d(

199

Hg). In

bis-ﬂuoroalkyl mercury derivatives

199

Hg,

F) de-

creases as the donor ability of the solvent increases.

In CdMe

113

H) is positive, but

113

has the opposite sign. Although

199

H) is

negative in alkyl derivatives of mercury it is positive
in vinyl derivatives. Couplings to

F in Hg–CF

compounds have the opposite sign to those to

H in

the analogous Hg–Me system.

See also: Nuclear Magnetic Resonance Spectroscopy:
Overview; Principles. Nuclear Magnetic Resonance
Spectroscopy-Applicable

Elements:

Hydrogen

Iso-

topes; Carbon-13; Fluorine-19; Nitrogen-15; Phosphorus-
31. Nuclear Magnetic Resonance Spectroscopy Ap-
plications: Proton NMR in Biological Objects Subjected
to Magic Angle Spinning. Nuclear Magnetic Resonance
Spectroscopy Techniques: Solid-State; Surface Coil.

Further Reading

Gielen M, Willem R, and Wrackmeyer B (eds.) (1997)

Advanced Applications of NMR to organometallic
Chemistry, Physical Organometallic Chemistry, vol. 1.
New York: Wiley.

Kidd RG (1971) NMR spectroscopy of organometallic

chemistry. In: Tsutsui (ed.) Characterization of Organo-
metallic Compounds, part II, ch. 8. New York: Wiley-
Interscience.

Mann BE (1974)

C NMR chemical shifts and coupling

constant of organometallic compounds. Advances in
Organometallic Chemistry 12: 135–213.

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-
tons, ch. 11: 153–156. New York: Wiley-Interscience.

Mann BE (1988) Recent developments in NMR spectro-

scopy of organometallic compounds. Advances in Organo-
metallic Chemistry 28: 397–457.

Mann BE, et al. (1985) Journal of the Chemicl Society,

Dalton Transactions: 693.

Orrell K (1999) Dynamic NMR spectroscopy in inorganic

and organometallic chemistry. Annual Reports on NMR
Spectroscopy 37: 1–74.

Roc (1987) CD spectroscopic characterization of inorganic

and organometallic complexes by metal and high-pres-
sure NMR. In: Wayda AL and Darensbourg MV (eds.)
Experimental Organometallic Chemistry, ACS Symposi-
um Series No. 357, ch. 8. Washington, DC: American
Chemical Society.

Vrieze K and Van Leeuwen PWNM (1971) Studies of dyna-

mic organometallic compounds of the transition metals
by means of NMR. Progress in Inorganic Chemistry 14:
1–63. New York: Wiley Interscience.

NUCLEAR MAGNETIC RESONANCE
SPECTROSCOPY APPLICATIONS

Contents

Food

Forensic

Pharmaceutical

Proton NMR in Biological Objects Subjected to Magic Angle Spinning

Food

C Deleanu

, National NMR Laboratory and Institute of

Organic Chemistry, Bucharest, Romania

Introduction

Food materials exhibit a wide range of states of
complexity. These range from homogeneous and re-
latively simple mixtures, as in edible oils, to quite
complex heterogeneous systems, as in meat or bread
(in the latter case both air and heterogeneous solid

Table 19

Ranges of some spin–spin coupling constants for Cd

and Hg organometallic compounds

Complex type/ligands

Range (Hz)

113

Cd,

Alkyls

49–90

113

Cd,

Alkyls, CN

271–1060

113

Cd,

Alkyls

6–70

113

Cd,

Alkyls

17–45

199

Hg,

Alkyls

94–265

199

Hg,

Organomercurials

38–311

199

Hg,

Alkyls, aryls, CN

264–3875

199

Hg,

Alkyls, aryls

24–920

199

Hg,

Alkyls, aryls

93–205

199

Hg,

Vinyls, alkyls

338–1911

199

Hg,

1980–12 970

NMR SPECTROSCOPY APPLICATIONS

/ Food

303

phases are present). This range of physical states
and chemical compositions requires a wide palette of
nuclear magnetic resonance (NMR) techniques and
approaches for obtaining in-depth information and
characterization of various foodstuffs. NMR tech-
niques are versatile and can provide different types
of data and information on the same sample, depen-
ding on the experimental parameters selected. The
applications include qualitative and quantitative
analysis monitoring of processes or biochemical re-
actions determination of the structure of isolated
compounds, the mobility and state of fat and water,
the microstructure and aggregation state of various
components in foodstuffs, as well as macroscopic
imaging with all the advantages of obtaining cross
sections without the need for cutting the sample. In
terms of quantifying various compounds, NMR
spectroscopy has the ability to provide the global
concentration of the sample and not only the surface
concentration. The disadvantages of the early days
of NMR spectroscopy such as ‘low sensitivity’ or
‘expensiveness’ of the instrumentation do not hold
true in the early twenty-ﬁrst century. Thus, the mod-
ern hardware, software, automation, and hyphena-
tion have reduced the experimental time to a few
minutes even for two-dimensional (2D) experiments
and increased the sensitivity, allowing easy detection
of compounds at levels of micrograms and even
nanograms. Moreover, there are arguments sup-
porting the fact that sometimes well established
techniques like gas chromatography (GC)–mass
spectrometry (MS) produce false results, several of
the ‘new’ compounds reported based on these tech-
niques being in fact experimental artifacts resulting
from the decomposition of some original labile com-
pounds in foodstuffs. In such cases NMR has the
great advantage of providing information on the
studied compounds under ambient conditions of
temperature and pressure.

High Resolution (HR) NMR (Frequency
Domain NMR)

Most HR NMR food sciences analyses are done in
solution. However, the information provided by HR
solid-state NMR is also very valuable for food sci-
ences. Thus it is very likely that in the near future we
will see a more balanced number of liquid- and solid-
state NMR studies in food sciences.

Elucidating the Structure of Isolated Compounds

HR NMR is currently the most powerful technique
for elucidating the structure of isolated compounds
in solution. There are numerous papers describing

the determination of the structure of various com-
pounds isolated from all types of foodstuff. This ap-
plication is an essential tool in advanced food
research and requires prior isolation and puriﬁca-
tion of the compound under study.

As this approach is identical to that employed in

synthetic chemistry, we would not cover it in this
article. The interested reader will ﬁnd extensive
coverage of NMR techniques for structure determi-
nation in other articles of this encyclopedia.

The following sections will provide examples of

applications of various NMR techniques to mixtures
of compounds in various food matrices or ﬂuids.

Fruits and Fruit Juices

H-NMR spectra for various fruit juices have been

published, and signals have been assigned to sugars,
amino acids, and other compounds like alcohols, ac-
ids, and polyphenols. Furthermore,

H-NMR spectra

have been used in combination with chemometric
techniques for discriminating between apple varieties
and for detecting adulteration of orange and apple
juices. There are several non-NMR methods classi-
cally employed for determination of fruit juice adul-
teration (density, total amino acids, ﬂavonoid and
carotenoid composition). Some of these methods
(e.g., liquid chromatography (LC)) require however
standards of the suspected markers that are not
always available. Moreover, with the classical meth-
ods one should preselect the most likely markers for
a particular sample based on an educated guess on
the expected type of adulteration. HR NMR has the
advantage of offering a global proﬁle. Thus, even
without a total assignment of signals, in conjunction
with chemometric techniques the method can dis-
criminate between various patterns.

Figure 1 shows examples of

H-NMR spectra for

some common fresh fruit juices.

The typical proﬁle of the

H-NMR spectrum of

fruits and fruit juices could be rationalized by con-
sidering three regions. Between 0.5 and 3 ppm there
are various signals generated by amino acids, other
carboxylic acids, and alcohols. Between 3 and
5.5 ppm the NMR spectrum is dominated by sugars
as well as by the residual water signal (4.8 ppm).
Although other compounds give rise to some signals
in this region, in most cases these signals are of little
practical use as they are overlapped by the sugar
signals. The region between 5.5 and 10 ppm exhibits
signals generated by aromatic and heterocyclic com-
pounds, formic acid, and aldehydes.

Solid-state

C-NMR has also been used to study

fruits, for instance to follow changes in the cell wall
polymers during ripening.

304

NMR SPECTROSCOPY APPLICATIONS

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7.5

7.0

6.5

ppm

7.5

7.0

6.5

ppm

7.5

7.0

6.5

ppm

7.5

7.0

6.5

ppm

7.5

7.0

6.5

ppm

(A)

(B)

(C)

(D)

(E)

ppm

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.

NMR SPECTROSCOPY APPLICATIONS

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305

Vegetables

The assignment of many components in the

H-NMR

spectrum of tomato juice has been published recently.
The

H-NMR proﬁle of tomato juice resembles that

of fruit juices, with the same distinct zones, i.e.,
aminoacids, sacharides, and aromatic compounds.

H-NMR spectra of methanolic extracts of control

and genetically modiﬁed varietals of tomatoes have
been analyzed using chemometric techniques. It was
possible to assess variations in several metabolites.

H, and

C solid-state magic angle spinning

(MAS) NMR spectra have been recorded for tomato
skin and potato tissue, providing information on the
structure and dynamics of the cuticle polyesters.

It has been demonstrated that for the case of some

ﬂavor compounds present in garlic, NMR is superior
to GC–MS as the latter produces false results due to
the decomposition of some labile compounds.

Large differences in the ratio between proteins

and polysaccharides in various mushroom strains
have been observed using

C solid-state MAS NMR

spectroscopy.

Wine

Surprisingly, in contrast with the early success of

H-NMR spectroscopy in wine analysis (see Wine

section of SNIF-NMR),

H-NMR has not been used

on whole wine samples until very recently. Although
the possibility of quantifying methanol in wine was
proved in the early 1990s, it was only a decade later
that the ﬁeld took off, with many minor compounds
identiﬁed in whole wine samples and with chemo-
metrics helping to rationalize the global proﬁle of the

H-NMR spectrum of wine samples. The same trend

is paralleled with spectra for other alcoholic beve-
rages like beer.

Recently an interesting application used a home-

made spectrometer based on a 310 mm bore magne-
tic resonance imaging (MRI) instrument operating at
2 T to record

H- and

C-NMR spectra of intact

unopened wine bottles. Thus, NMR was able to
detect spoiled bottles with acetic acid developed in
them without opening them. The application could
be very valuable for old and expensive wines.

Sugars (Carbohydrates)

There are many studies involving

H- and

C-NMR

spectroscopy of carrageenans, a family of poly-
saccharides with the structure of linear sulfated
galactans. They are extracted from certain species
of red seaweed and have been used for a long time as
natural texturing ingredients in the food industry.

The polysaccharide b-glucan has been proved to

induce beneﬁcial effects such as immunobiological,

hypocholesterolemic,

and

hypoglycemic

effects.

-Glucan is isolated from yeast, and the established

wet analytical methods are quite tedious.

H-NMR

has been proved to be a rapid alternative method.

Sugars have been determined using both liquid-

and solid-state NMR in fruits, vegetables, and their
juices, as well as in honey.

Starch products and glycogen have been studied

extensively using

H-,

C-, and

P-NMR spectros-

copy. NMR spectroscopy has been used for determi-
ning the degree of polymerization, the average
number of glucose units, the branching degree, and
the anomer distribution of the reducing sugars. The
interaction of starch with water and the mobility of
polysaccharide

chains

have

been

investigated

through one-dimensional (1D) and 2D solid-state
NMR experiments. The mobility of ‘freezable’ and
‘unfreezable’ water in starch was studied using

and

H solid-state NMR. The aging (retrogradation)

of starch has been studied using cross polarization
magic angle spinning (CPMAS)

C-NMR.

Figure 2 presents the

H-NMR spectrum of

glucose. As always, in solution glucose is formed by
a mixture of a- and b-anomers. The spectrum em-
phasizes the region between 3.2 and 4.0 ppm, which
is very crowded in the case of saccharides, glucose
being only one of the saccharides present in various
foodstuffs. Thus, many food products (including
fruits, vegetables, honey, and wines) exhibit an NMR
spectrum crowded with signals in the saccharide
region.

Tea

Several ﬂavonoids isolated from tea have been analy-
zed and their structure determined using NMR.
There are several problems with the classical meth-
ods of analysis of ﬂavonoids in tea. Due to the pres-
ence of complex mixtures of ﬂavonoids in tea, they
are often characterized as ‘total polyphenols’. The
colorimetric method for analysis of total phenols can
interfere with other reducing compounds. LC can
well resolve peaks for individual ﬂavonoids; how-
ever, there are only a few standards available com-
mercially, making the assignment of peaks uncertain
in many cases. Thus, the structure of ﬂavonoids
giving rise to peaks in LC is often determined using
various 1D- and 2D-NMR experiments.

Coffee

The early NMR studies of coffee were limited to
elucidating the structure of isolated compounds.

Several of the major constituents of espresso coffee

have been identiﬁed in the

H-NMR spectrum.

H-NMR data in combination with chemometrics

306

NMR SPECTROSCOPY APPLICATIONS

/ Food

have been recently used for verifying the authenticity
of instant coffees from various producers.

Milk and Dairy Products

The water–methanol extracts of cheese have been
analyzed using

H-NMR, and assignments of several

amino acids were done using 2D spectra. Variations
in the content of amino acids were observed as a
function of ripening time and distance from the cen-
ter of the cheese wheel. The fatty acid triglycerides
from milk have been analyzed using both

H- and

C-NMR.

Liquid state

P-NMR has been employed since a

long time back for studying milk and casein. Solid-
state

P-NMR has been used for studying the nature

of micelles and the interaction between compounds
that form them.

Cereals and Bread

Solution-state

C-NMR and

P-NMR have been

employed in the study of lipids from corn suspension
cells. Corn cells have also been grown in the presence
of

C-enriched acetate for following its incorpora-

tion into oleates and linoleates.

Solid-state CPMAS

C-NMR has been used for

investigating changes in maize and sorghum proteins
on wet cooking and popping.

The hydration of proteins from wheat seeds has

been studied using solid-state

C CPMAS and

high resolution magic angle spinning (HRMAS)
NMR spectroscopy.

Fats and Oils

Edible fats and oils are ideal candidates for HR
NMR. Their good solubility in organic solvents
makes the NMR analysis fast, easy, and accurate.
Issues such as the composition of fatty acids in oils
and fats, adulteration with cheaper oils (especially in
the case of extra virgin olive oil), degradation of oils
and fats in time and at elevated temperatures, and
content of fatty acids in single intact seeds (for
breeding purposes) have been studied using NMR.
NMR method has some advantages over other meth-
ods. Thus, there is no need for sample manipulation
before the analysis, information on the distribution
of the fatty acid chains in the glycerol moiety (po-
sition 1/3 versus 2) is available, and the analysis has a
relatively high speed (even for

C-NMR, results

being obtained within minutes).

A typical

H-NMR spectrum for sunﬂower oil is

presented in Figure 3.

A typical

C-NMR spectrum for the same

sunﬂower oil is presented in Figure 4.

Figure 5 presents details of the region 172–

174 ppm in the

C-NMR spectrum (100 MHz),

illustrating the power of the method in assigning
the distribution of fatty acids in the positions 1/3 and
2 of the glycerol moiety, based on the signals of the
CO groups.

5.4

5.2

5.0

4.8

4.6

4.4

4.2

4.0

3.8

3.6

3.4

ppm

Figure 2

The

H-NMR spectrum of a mixture of a- and b-glucose in D

O recorded at 400 MHz. The high peak at 4.8 ppm is the

residual water signal.

NMR SPECTROSCOPY APPLICATIONS

/ Food

307

If for most food applications, recording the NMR

spectra at the highest available magnetic ﬁeld
strength is a great advantage, for edible oils and fats
there is not much improvement on passing from 300
to 600 MHz. In this case, the same types of groups
from different long chain fatty acids produce similar
signals in the

H-NMR spectrum. This fact makes

the routine medium-ﬁeld (300–400 MHz) NMR
spectrometers suitable and very competitive (in terms
of price-to-quality ratio) for research and quality
control of edible oils.

Individual fatty acids and esters have been used as

model compounds for assignment of signals in the

NMR spectra of edible oils and fats. The concentra-
tion dependence on chemical shifts of model triglyce-
rides has also been explored. Several papers deal with
authentic edible oil samples, with reference to both
assignment of NMR signals and authentication is-
sues. As expected, many papers deal with NMR of
olive oils due to the high market value of the virgin
and extra virgin qualities. A lot of effort is put in
authentication and determination of the origin of
olive oils in connection with the Denomination of
Protected Origin. Papers also deal with the identiﬁ-
cation of mono- and diglycerides in edible oils, as
well as other compounds like phenolic derivatives.

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

ppm

Figure 3

H-NMR spectrum (400 MHz, CDCl

) of a sample of sunﬂower oil.

160

140

120

100

ppm

Figure 4

C-NMR spectrum (100 MHz, CDCl

) of a sample of sunﬂower oil.

308

NMR SPECTROSCOPY APPLICATIONS

/ Food

Polyunsaturated fatty acids (PUFAs), characteristic of
ﬁsh lipids but also present in small quantities in
vegetable oils, have been also investigated using
NMR. Apart from the composition in fatty acids or
minor compounds, the CH region in

H-NMR, and

the CH

region in

C-NMR have been used for de-

termining the iodine value. Several studies deal with
issues like the changes induced in the composition of
oils by oxidation, heating, or hydrogenation.

Seeds

C-NMR spectra of intact seeds allow the identiﬁ-

cation and quantitation of the main fatty acids.

Solid-state

C HRMAS and distortionless en-

hancement by polarization transfer (DEPT) MAS
NMR also have been used for studying integral
seeds. Thus, the three major components, fats, carbo-
hydrates, and proteins (including the position of
the fatty acid in glycerol) could be identiﬁed and
quantiﬁed.

In vivo

P-NMR has been used for studying the

metabolites during the ripening and drying of seeds.

Fish

Fish oils have been extensively investigated using

H-

and

C-NMR. PUFAs, important in diet, are be-

lieved to reduce the risk of cardiovascular diseases.
NMR analysis of PUFA is an interesting alternative
to the widely used GC and GC–MS techniques when
artifacts may arise either during transesteriﬁcation
or within the GC–MS equipment due to elevated

temperatures. PUFAs are markers for the ﬁsh species,
age, freshness, storage, and processing conditions.

Meat

C solid-state HRMAS NMR studies have been car-

ried out on cartilages.

P-NMR spectroscopy studies of energy-related

metabolites have been carried both in HR NMR
instruments as biopsies and in MRI instruments as
entire muscle pieces. The phosphorus metabolites are
directly linked to meat quality. The inﬂuence of
the stunning method on the postmortem energy me-
tabolism and meat quality has been explored
using

P-NMR. The pH has been determined using

P-NMR and associated with the meat quality.

Although the pH is measured routinely through
voltammetry, the NMR method has the advantage of
measuring the pH in an inner part of the sample,
being able to assess it both intra- and extracellulary,
as well as in vivo.

Eggs

The oxidation of cholesterol in egg powder has been
detected using

H-NMR.

P-NMR spectroscopy has

been used to quantitate phospholipids in egg lecithin.

Essential Oils

Essential oils are important ﬂavors and aromas.
Many individual compounds have been isolated and
characterized. Unseparated mixtures forming essen-
tial oils have also been analyzed using

C-NMR.

173.1

173.0

172.9

172.8

172.7

ppm

Saturated
acids

Oleic
acid

Linoleic
acid

1/3

Linoleic
acid

Oleic
acid

Figure 5

Enlargement of the carbonyl region in the

C-NMR spectrum (100 MHz, CDCl

) of a sample of sunﬂower oil.

NMR SPECTROSCOPY APPLICATIONS

/ Food

309

Agricultural Chemistry

It has been shown that even with medium-ﬁeld NMR
spectrometers (300 MHz), quantitative

H- and

P-NMR can rival with chromatography as an

analytical method for agricultural chemicals. The
advantage of the NMR is that it does not require a
reference standard as is the case with chromato-
graphy. The possibility of detecting organophospho-
rus insecticide residues in crops using

P-NMR has

been demonstrated. Soil samples have been analyzed
using

C, and

P solid-state and solution-state

NMR experiments.

Plant and Cell Metabolism and
Other Biotransformation

The metabolism of plants has been followed ex-
tensively using various NMR techniques, employing

P, and

C nuclei. All kinds of samples, cells,

tissues, and extracts, both in vivo and in vitro, have
been investigated. A special hardware design is used
for following biotransformations in vivo. Thus the
living material is suspended in a culture medium in
the NMR tube and air is bubbled through a capillary
tube to maintain life.

H-NMR (Site Specific Natural

Isotope Fractionation Using NMR
(SNIF-NMR))

H SNIF-NMR is a high-resolution technique.

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.

Wine

Wine analysis using

H-NMR is one of the major

applications of NMR in food sciences, probably the
most important application of HR NMR.

The basis for authentication of the geographic

origin of wine and for spotting its adulteration is
deuterium (

H) NMR spectroscopy. The method

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
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 CH

and CH

groups of the ethanol molecule in the

H-NMR

spectrum. The method requires prior distillation of
the ethanol from wine. The power of the method
relies on the fact that the

H ratio in various

positions of an organic molecule depends on factors
such as the plant metabolism (thus bearing infor-
mation on the type of sugar that by fermentation
produces the ethanol) and the isotopic composition
of the ground and rain water (thus bearing infor-
mation on the geographic position). The isotopic
ratio

H in water varies on Earth between 90

and 160 ppm, depending on the latitude, with the
highest values at the equator. It has been proven
that deuterium in the CH

site of the ethanol

molecule comes from sugars, whereas deuterium
in the CH

site comes mainly from water. Thus, in

the end, the isotopic ratio

H in different sites

of the distilled ethanol bears complex information
related to the grape type, geographical origin, cli-
mate, production year, added sugar, or added
water. In order to improve the power of the meth-
od, the isotopic composition in the two sites is no
longer expressed as a relative ratio but as an abso-
lute ratio, calibrated against a standard of known
isotopic composition. The currently ofﬁcial standard
is N,N-tetramethylurea (TMU). Typically ﬁelds of

(A)

(B)

(C)

TMU

ppm

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

ppm

Figure 6

HR NMR spectra of ethanol recorded at 9.4 T. (A)

H-

NMR (61 MHz); (B)

H-NMR (400 MHz); and (C)

C-NMR

(100 MHz).

310

NMR SPECTROSCOPY APPLICATIONS

/ Food

400 or 500 MHz are used in routine analysis;
however, owing to the good separation of deute-
rium signals, 300 MHz instruments can equally
provide reliable results.

Figure 6 presents the

H-NMR spectrum of et-

hanol, employed in the SNIF-NMR method, and for
comparison the

H- and

C-NMR spectra.

SNIF-NMR is also applied to a whole range of

alcoholic beverages, including: cognac, whisky, beer,
and grape must.

Fruit Juices

SNIF-NMR is already a well established technique
for fruit juices, being suitable for tracing both their
geographical origin and adulteration. The method
was originally based exclusively on

H analysis,

but later its discrimination power was greatly in-
creased by using it in combination with

analysis through isotopic ratio mass spectrometry
(IRMS). In the case of fruit juices SNIF-NMR is
mostly used for detecting adulteration with cheaper
sugars, being accepted as an Association of Ofﬁcial
Analytical Chemists (AOAC) method.

Miscellaneous Applications of SNIF-NMR

Apart form wine and juices the method has been suc-
cessfully applied to issues such as the authentication
of sugars of various origins, origin of glycerol (sugar
fermentation versus edible oil transesteriﬁcation),

natural versus synthetic origin of aromas, vari-
ous essential oils, terpenoids, and others. There is a
clear legislative discrimination between ‘chemically
identical’ and ‘natural’ food aromas and ﬂavors, the
‘natural’ ones being premium products. Owing to
the great difference in price between such natural and
synthetic aromas, the adulteration techniques be-
came more and more sophisticated. Thus, vanilla
adulteration techniques included even the addition
of

C- and

H-enriched vanillin. Consequently, the

analytical methods have had to be improved over
the years, and presently combinations of

H SNIF-

NMR,

C IRMS, and chemometrics applied to

several marker compounds in the ﬂavor can success-
fully cope with the most sophisticated types of adul-
teration used to date.

Hyphenated Techniques

Emerging

hyphenated techniques have already

proved their analytical value and in the near future
will certainly become common techniques in food
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-
ly hyphenation with solid-phase extraction (LC–solid
phase extraction–NMR).

Figure 7 illustrates the power of the on-ﬂow liquid

chromatography (LC)–NMR technique in the ana-
lysis of grape juice.

RT 26 min

RT (min)

RT 16 min

RT 13 min

RT 6 min

ppm

6.5

7.0

7.5

8.0

ppm

6.5

7.0

7.5

8.0

6.0

6.5

7.0

7.5

(A)

(C)

(B)

8.0

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.)

NMR SPECTROSCOPY APPLICATIONS

/ Food

311

Low-Resolution (LR) NMR (Time
Domain NMR)

LR-NMR is the most commonly used NMR tech-
nique to date for quality control in food science and
industry, with several ofﬁcial quality control methods
in force. The success of the technique is due to
several factors, including the power of the method (in
terms of information and speed), the early applica-
tion of the method to foodstuffs, the ecological ap-
peal (no longer needing polluting chemicals), and the
relatively low cost of the equipment, making it a very
attractive alternative to the tedious wet chemical
methods.

Fats and Oils

Solid fat content (SFC) analysis is probably the most
used LR-NMR application in the food industry. The
initial success of the method prompted Unilever
(manufacturer of margarines and related products)
and Bruker (manufacturer of NMR instruments) to
start a joint venture with the goal of building a table-
top LR-NMR spectrometer for solid-to-liquid ratio
analysis in the fat industry. The method was
developed in the early 1970s and over the years
leaded to various quality control protocols for fat
and oils that are by now adopted as ofﬁcial methods
by various international and national organizations.
The success story of SFC analysis opened wide the
door for LR NMR methods to penetrate as routine
techniques in the food industry.

As the relaxation time depends on the mobility of a

particular system (with solids having the shortest re-
laxation times), it is easy to quantify mixtures using
NMR. The bulk magnetization decay (free induction
decay (FID)) for samples containing various solid-to-
liquid ratios is presented in Figure 8.

The SFC is a critical parameter for the fats and oils

industry. The ofﬁcial American Oil Chemists’ Society
(AOCS) wet method is dilatometery. Alternative wet
methods are differential thermal analysis and differ-
ential scanning calorimetry (DSC). LR NMR was
proved to be an alternative method for SFC determi-
nation in late 1950s. The early continuous wave LR
NMR spectrometers rapidly found their way into the
fats and oils industry, the method being accepted by
the Instrumental Techniques Committee of the AOCS
as early as in 1972. Presently the technical choice is
radio frequency (RF) pulsed LR NMR. Pulse NMR
spectrometers are more compact, very efﬁcient, and
relatively cheap. They have the advantage of exciting
the protons in the whole sample at once.

The determination of SFC has been extended to

oil-in-water emulsions, where additional experimen-
tal parameters have to be considered in order to dis-
tinguish between oil and water protons. The droplet
size of oil-in-water emulsions is usually measured
using optical diffraction techniques, but it has also
been determined through diffusion NMR experi-
ments.

The degradation of fats and oils during frying has

also been determined using LR NMR. During frying
oils undergo complex chemical reactions such as
oxidation, polymerization, hydrolysis, and isomeri-
zation. During these processes polar groups accumu-
late. The ofﬁcial International Union of Pure and
Applied Chemistry and AOAC method for assessing
the frying fat quality is the determination of total
polar groups through preparative column chro-
matography. Based on the fact that the relaxation
time is decreasing in direct relationship with the
amount of polar groups present in the sample, it has
been proved that LR NMR data correlate well with
the values obtained using the ofﬁcial column chro-
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.

Seeds

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
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
capacity of oil production.

The principle of separating and quantifying vari-

ous components in a system based on the T

relax-

ation time is the following. Various components have
various relaxation times mainly due to differences in

0% liquid + 100% solid

Time

Amplitude

30% liquid + 70% solid

70% liquid + 30% solid

100% liquid + 0% solid

Figure 8

Bulk NMR signal amplitude for samples with various

solid-to-liquid ratios. The signal decays exponentially with a con-
stant T

relaxation time.

312

NMR SPECTROSCOPY APPLICATIONS

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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
(FID). Water has an intermediate relaxation time,
whereas oil has the longest relaxation time. Thus, by
carefully selecting the region of the FID where the
component of interest is making the major contribu-
tion, one can quantify this component relative to
other components. Meanwhile the intensity of the
signal is proportional to the number of nuclei (pro-
tons) generating that signal. Thus by comparing the
intensity of FID in the region of the component of
interest for different samples or in comparison with a
calibrated standard, one can obtain quantitative re-
sults. Figure 9 illustrates how water in various seeds
can be quantiﬁed with LR NMR when analyzing the
middle part of the FID signal. Thus, by drying seeds
at room temperature, the quantity of water that re-
mains is higher (and the NMR signal’s amplitude is
higher) than by drying the same seeds at 60 or 100

1C.

An almost identical curve was obtained on drying

various seeds (peanut kernels, mustard, sunﬂower,
and soybean) by sun and at 105

1C.

When it is desired to quantify the slowly relaxing

oil component, a more suitable pulse sequence
scheme would be the spin-echo. As illustrated in
Figure 10, the time at which the second refocusing
pulse (of 180

1) is applied can be selected so as to

obtain an NMR signal generated exclusively by oil.
The oil content can be accurately quantiﬁed when the
amplitude of the signal is compared with a calibra-
tion curve generated by standard samples.

Fruits

LR NMR has been used to determine the changes in
water mobility in dehydrated fruits, and the sugar

content in intact fruits. The technique is suitable for
following the ripening process.

Sugars (Carbohydrates) and Starch

LR NMR is a routinely used technique for determi-
nation of water content in sugars.

Starch hydration, water mobility, and the effect of

chemical modiﬁcation on molecular mobility of
starch have also been studied using LR NMR.

Alteration of starch over time (retrogradation) has

been studied using LR NMR. Most of the studies
employed the T

relaxation curves of

H-NMR.

Thus it was shown that the number of protons in the
solid phase increases with aging, this being explained
by the recrystallization of starch.

Milk and Dairy Products

The state of water in milk, milk powder, casein, whey
proteins, and cheese has been investigated using LR
NMR.

Chocolate

LR techniques are routinely used for determination
of the fat content and solid-to-liquid ratio in choco-
late and cocoa products. Analysis of the ﬂuidiﬁca-
tion of cocoa butter using LR NMR enabled the
identiﬁcation of cocoas according to the process and
the type of roaster used.

Amplitude

Oil

Time

(C)

(B)

(A)

Moisture

and oil

Proteins,

sugars,

moisture,

and

oil

Figure 9

Effect of different drying methods on FID from sun-

ﬂower seeds. The ﬁnal moisture content is different when drying
takes place at 100

1C (A), 601C (B), or room temperature (C).

Water

Oil

Time

180

Time

(B)

Amplitude

(A)

Figure 10

The spin-echo RF pulse sequence (A) and its result

on the amplitude of the NMR signal (B) when applied to corn
samples.

NMR SPECTROSCOPY APPLICATIONS

/ Food

313

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
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 T

(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 T

LR NMR of water based on different

types of collagen present in various types of tissue.
Another important parameter of meat is the
development of ﬂavor associated with a long storage
time

and

storage

temperature.

The

so-called

warmed-over ﬂavor (WOF) is produced by autoxi-
dation of membrane phospholipids and degradation
of proteins and heteroatomic compounds. Some
WOFs have been predicted using LR NMR and
chemometrics.

Bread

LR NMR can monitor the distribution and mobility
of water during bread making (dough, baking to
bread, staling).

The baking process has been performed inside the

NMR magnet and followed using the

H T

relax-

ation time. The water mobility during storage of
bread has been studied using the T

and T

relaxa-

tion curves. The texture parameters (elasticity and
ﬁrmness) have correlated well with T

curves of

bread crumbs.

Potatoes

Prediction of the sensory texture properties of
cooked potatoes, based on raw potato analysis, is
of major importance to the food industry. The sen-
sory texture quality of potatoes has been predicted
using T

LR NMR. Correlation of the T

LR NMR

data with the chemical composition of potatoes has
also been performed using chemometrics. Differen-
tiation between potato varieties and determination of
dry matter content has also been done using T

and

LR NMR.

NMR Imaging (Spatial Domain NMR)

The current MRI techniques provide information not
only on the internal structure of various foodstuffs
but also on the distribution of various compounds
(chemical shift imaging), mobility and diffusion of
various compounds (like water, oil, or compounds
from various packing materials), or dynamic changes
like crystallization.

The technique is increasingly being used in food

sciences but still less than its true potential. The ex-
planation for the delay in widespread use of NMR
imaging techniques in food science and industry is
the high cost of the equipment.

Figure 11 shows four MRI cross-sections through

a kumquat fruit. They start from near the surface
and advance toward the center of the fruit. The fruit
ﬂesh in the centre is surrounded by the peel. The
tissue between the fruit ﬂesh and the peel is not
visible because of a very short T

relaxation time.

Spherical structures in the peel are visible, and they
contain the typical aromatic contributions of the
fruit.

MRI has been applied to various foodstuffs such as

meat, cereals, seeds, fruits, vegetables, cheese, or
chocolate. The distribution and mobility of various
constituents including water, sugars, and lipids have
been monitored.

See also: Carbohydrates: Overview; Sugars – Spectro-
photometric Methods. Food and Nutritional Analysis:
Overview; Coffee, Cocoa, and Tea; Alcoholic Beverages;

Figure 11

MRI cross-sections through a kumquat fruit at 7 T.

(Reproduced with permission from Bruker BioSpin, Rheinstetten,
Germany.)

314

NMR SPECTROSCOPY APPLICATIONS

/ Food

Meat and Meat Products; Dairy Products; Vegetables and
Legumes; Oils and Fats; Fruits and Fruit Products. Lip-
ids: Overview; Fatty Acids. Liquid Chromatography:
Liquid Chromatography–Nuclear Magnetic Resonance
Spectrometry. Mass Spectrometry: Overview. Nuclear
Magnetic Resonance Spectroscopy: Overview; Princi-
ples; Instrumentation. Nuclear Magnetic Resonance
Spectroscopy-Applicable Elements: Hydrogen Iso-
topes; Carbon-13. Nuclear Magnetic Resonance Spec-
troscopy Techniques: Solid-State; In Vivo Spectroscopy
Using Localization Techniques.