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TD-NMR in Quality Control: Standard Applications Advertisement

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DOI: 10.1007/978-3-319-28275-6_12-1
In book: Modern Magnetic Resonance, pp.1-18
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Marcio Fernando Cobo

Eleonore J. Deublein
14.99 · Bruker Corporation

Agnes Haber Rance Kwamen

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22.56 · Bruker Corporation 9.74

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Abstract

This chapter is focusing on Time-Domain Nuclear Magnetic Resonance (TD-NMR) relaxometry and diffusometry methods and their use in food
development, process, production, and quality control (QC). The widely used standard applications and their variations to determine solid fat content
(SFC), droplet size distribution, and oil and moisture contents in food using TD-NMR technology will be discussed. In addition, this chapter will discuss
the use of automation for industrial applications to ease the handling and maximize the analysis throughput. This chapter is organized in five main
sections, starting with introduction; discussing SFC, droplet size determination, and oil and moisture applications; and later concluding the chapter.

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One pulse NMR experiment, where Pulse sequence used for measuring Application areas of TD-NMR for G-Var signal acquired as function of
the signal from the combined liqui… droplet size distributions: PFG-SE… the determination of oil and… the gradient strength (T/m)

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TD-NMR in Quality Control: Standard

Applications

Marcio Fernando Cobo, Eleonore J. Deublein, Agnes Haber,

Rance Kwamen, Manoj Nimbalkar, and Frank Decker

Abstract
This chapter is focusing on Time-Domain Nuclear Magnetic Resonance
(TD-NMR) relaxometry and diffusometry methods and their use in food devel-
opment, process, production, and quality control (QC). The widely used standard
applications and their variations to determine solid fat content (SFC), droplet size
distribution, and oil and moisture contents in food using TD-NMR technology
will be discussed. In addition, this chapter will discuss the use of automation for
industrial applications to ease the handling and maximize the analysis throughput.
This chapter is organized in five main sections, starting with introduction;
discussing SFC, droplet size determination, and oil and moisture applications;
and later concluding the chapter.

Keywords
TD-NMR • Solid Fat Content (SFC) • International Standard Methods • Food
quality control • Droplet size distributions • Oil and moisture • Combined
relaxation analysis • AOCS Official Method Cd 16b-93 • Direct method • Indirect
method • ISO 8292 • IUPAC 2.150 • Fully automated SFC analysis systems •
PFG-SE • G-Var

Marcio Fernando Cobo, Eleonore Deublein, Agnes Haber, Rance Kwamen, Manoj Nimbalkar and
Frank Decker contributed equally to this work.
M.F. Cobo • E.J. Deublein • A. Haber (*) • R. Kwamen • M. Nimbalkar • F. Decker
Applied, Industrial & Clinical MR Division, Bruker BioSpin GmbH, Rheinstetten, Germany
e-mail: Marcio-Fernando.Cobo@bruker.com; Eleonore.Deublein@bruker.com; aicapps@bruker.
com; agnes.haber@bruker.com; Rance.Kwamen@bruker.com; Manoj.Nimbalkar@bruker.com;
Frank.Decker@bruker.com

# Springer International Publishing AG 2017 1

G.A. Webb (ed.), Modern Magnetic Resonance,
DOI 10.1007/978-3-319-28275-6_12-1

2 M.F. Cobo et al.

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Solid Fat Content (SFC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Direct Method (AOCS Official Method Cd 16b-93) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Indirect Method (AOCS Official Method Cd 16–81) . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 6
Automation System for SFC Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Conclusion . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Droplet Size Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Introduction . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 7
δ-Var and G-Var Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
G-Var NMR Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Calibration and Measurement G-Var Concept and Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0
Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Conclusion . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Oil and Moisture Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Introduction . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 12
Oil and Moisture Content Determination for Low Moisture Content Food .. . . . . . . . . . . . . . . . 1 3
Combined Relaxation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Conclusion . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6
References . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Introduction

Quality control (QC) is a very important topic in food development, process, and
production. Food industries must assure the high quality and safety of their products
in a fiercely competitive and regulated field. Therefore, they have implemented
various quality control measures at different stages of the development and process
from incoming material to outgoing products. Not only food industries but also
regulatory institutes around the globe seek to continuously improve the quality and
the robustness of these quality control measures, for example, but not limited to
doing comprehensive checks at every single processing and production step. This is
also important when it comes to cost-effectiveness of the production and the quality
control measures themselves. Various methods have been employed in the field of
food quality and process control starting from human sensory and classical chemical
analysis to various state-of-the-art applied analytical techniques. There is a range of
applied analytical techniques available to date such as ultraviolet spectroscopy,
atomic absorption spectroscopy, infrared spectroscopic, radiation spectroscopy,
and nuclear magnetic resonance (NMR) spectroscopy.
More than 45 years ago, the first benchtop TD-NMR systems have been used to
measure the so-called Solid Fat Content (SFC) in fat compositions. Since then, fat
industry as well as fat research is using this fast and simple method due to its
multiple advantages over alternative approaches for all types of fat and oil. Cocoa
butter, cocoa butter equivalents, as well as margarine fats or butter fats can be
accurately analyzed by TD-NMR. Apart from analyzing the samples according to

TD-NMR in Quality Control: Standard Applications 3

the International Standard Methods [1], many users are also interested in a quick
production check, thus measuring samples as they come from the line. The method
can easily be expanded to investigate SFC in samples with low amount of moisture
(<10%) like chocolate.
In the mid-1980s, water droplet sizing has become an established method in Food
QC laboratories as well as in product development environments. The motion of
molecules within the droplets phase of the emulsions is characterized by hindered
diffusion, thus allowing TD-NMR diffusion experiments to assess droplet size
distributions [2]. This provides information about shelf time and mouth feeling.
Typically, samples like margarines, diet spreads, and butter are examined by this
solvent-free approach. In 2001, the method could be expanded to oil droplet size
applications; therefore, the examination of samples like mayonnaise, salad dressing,
or soft cheese became possible. Droplet size methods have been improved in the past
few years, speeding up this approach easily by a factor of 3–5. Today a droplet size
measurement can be done in typically 3 min.
Shortly after the TD-NMR SFC method was introduced, oil and moisture deter-
mination in food samples became another QC standard [3]. This method uses the
Hahn-echo NMR pulse sequence, and soon this measurement became an Interna-
tional Standard Method. The only drawback of this method is that it can only
measure moisture/water content of up to 12% (depending on the sample type). If
the sample contains more than this amount, it has to be dried before the measure-
ment; otherwise, it gives erroneous values for both oil and moisture content. In order
to overcome this problem, a new combined relaxation analysis method was devel-
oped, enabling sample analysis with high water content.
This chapter will present a summary of the basic NMR techniques mentioned
above, namely, SFC, droplet size distribution, and oil and moisture analysis, with its
application in QC of food products.

Solid Fat Content (SFC)

The impact of the fat composition on the physical characteristics of fatty food is well
known. In the end product, the taste and color depend on the physical properties and
crystallization behavior of the fat phase [4]. Moreover, the properties of the fat have
to be taken into account during the manufacturing process when cooling or temper-
ing is necessary as this results in versatile consistency and masticatory melting
behaviors [5]. Therefore, several analytical methods have been developed for quality
control and assurance (QC/QA) in food industries.
TD-NMR is not only one of the internationally well-established solutions for
quality control in the industrial sector [6–8] but also an important tool in the
determination of Solid Fat Content for production and processing of fats and oils
in the food industry. TD-NMR has the following advantages: is noninvasive,
nonnecessity for additional chemicals, has good reproducibility and requires less
analysis time. Moreover, samples are easy to handle, and their preparation does not
require a high level of technical expertise as explained in the next sections.

4 M.F. Cobo et al.

By combining an NMR device with an automation system and cooling baths, sample
measurements can be ran continuously while the workflow is particularly facilitated
when following the various procedures and tempering schemes as described in the
International Standard Methods [1].
An alternative method that is traditionally used in the United States for charac-
terizing solid content of edible oils is to measure the solid fat index (SFI) by
dilatometry. The SFI method is based on the expansion of fat when heated. As
empirically observed, crystallized parts due to a higher chain ordering occupy less
space than liquefied fat that takes up more volume. Thus, the degree of expansion
can be correlated to the change of the solid/liquid ratio. Nevertheless, dilatometry
does not directly measure the solid content of fat; rather it measures the change in
volume compared to the starting point. Moreover, for samples containing emulsi-
fiers, the accuracy is questionable due to some dissolution of emulsifiers into the
indicator at the fat/indicator boundary.
The principle of SFC measurement by TD-NMR relies on detecting the 1H NMR
signal from both liquid and solid components in the fat sample (direct method) or by
detecting the change in the signal from the liquid phase as it is displaced by the solid
phase (indirect method). The measurement is based on the fact that the relaxation
process is strongly influenced by the interaction among neighboring nuclei. The
signal from molecules in the solid state decays faster than that from molecules in the
liquid state. Therefore, the NMR signal contains the analytical information about the
solid and liquid state in which the molecules exist. This results in a direct measure-
ment of the solids content that is highly reproducible and precise.
The first release of an AOCS official method for SFC determination by TD-NMR
was published in 1993 (AOCS Official Method Cd 16b-93) with the active contri-
bution of analytical companies such as Bruker. The current AOCS official methods
consist of “AOCS Cd 16b-93” revised in 2017 (also known as direct method) [9] and
“AOCS Cd 16–81” revised in 2017 (also known as indirect method) [10]. In Europe,
the official methods for SFC are the “ISO 8292” and “IUPAC 2.150” [1].

Direct Method (AOCS Official Method Cd 16b-93)

The direct SFC method is based on a one pulse NMR experiment. The free induction
decay (FID) that ensued from a single excitation pulse is recorded and further used to
determine separately the NMR signal arising from the sum of the solid and liquid
state and the liquid state alone. The contribution from the solid + liquid part is
measured at the shortest time allowed by the receiver hardware after the RF pulse
(= instrument dead time, less than 10 μs usually), while the contribution from the
liquid part alone is measured after the signal of the solid part is completely decayed.
This happens after 70 μs (Fig. 1), and the SFC value is computed using these two
measurements:

ðSSþL  SL Þ  f
SFCð%Þ ¼  100, (1)
ðSSþL  SL Þ  f þ SL

TD-NMR in Quality Control: Standard Applications 5

Fig. 1 One pulse NMR experiment, where the signal from the combined liquid and solid part is
acquired after 8.5 μs, while the liquid part contribution is recorded after 70 μs. The dotted line
represents the extrapolation of the FID to time zero by means of the f factor

where S S + L is the NMR signal due to both liquid and solid nuclei (recorded 8.5 μs
after the pulse) and SL is the NMR signal due to liquid nuclei (recorded 70 μs after
the pulse). The direct SFC method relies on a set of three calibration standard
samples for determining correction factors such as the f-factor that accounts for the
instrument dead time and corrects the amplitude of the solid fraction, the k-factor that
accounts for the effect of an additional signal decay due to magnet inhomogeneity,
and the o-factor that accounts for possible signal offsets.
In an industrial environment, the direct method is applied as a QC tool to control the
production of the fat compositions according to the desired properties. Thus, dedicated
protocols are followed. The sample preparation that consists of transferring the fat into
the NMR glass tube should preferably be done slightly (5  C) above the full melting
temperature of the fat in order to avoid any structural alteration. The sample filling
height should cover the entire sensitive volume of the NMR coil for a good sen-
sitivity. The tempering pathways depend on the needs and interest of the investigator.
While temperatures of 10, 21.1, 26.7, 33.3, and 37.8  C are used in the AOCS
Official Method Cd 16b-93, 10, 20, 25, 30, 35, and 40  C are used in the IUPAC
Standard Method. In the case of cocoa butter, SFC values between 10  C and 60  C
with 5  C increments are usually applied to obtain a higher resolution melting profile.
Regarding the tempering and sample handling procedures, two approaches, mentioned
below, are adapted, which do not necessarily provide the same SFC values.

Parallel Method
The parallel method uses at least one sample for each temperature to be measured
and is the fastest method to obtain the SFC values as a function of temperature. All
samples are crystallized at once at the same temperature (typically for 1 h).

6 M.F. Cobo et al.

Subsequently, the tubes are distributed over different tempering baths at different
temperatures. The TD-NMR measurement completes this parallel SFC method,
providing a good time shift planning during sample handling.

Serial Method
When the number of baths or amount of sample is limited, it may be necessary to
perform a serial setup. The serial method uses one sample which encompasses all
desired temperatures followed by the TD-NMR measurement, respectively. The
sample is crystallized at the desired temperature and then inserted into the first
tempering bath. After the tempering time has passed and the first NMR measurement
has been performed, the same tube is directly inserted into the next tempering bath
with the next measurement temperature, and so on. Due to the fact that the Solid Fat
Content is influenced by the thermal history of the sample, serial and parallel
measurements may lead to different results.

Indirect Method (AOCS Official Method Cd 16–81)

Though the direct method is well suited for QC due to the celerity in getting QC
parameters, one has to assume that the average f-factor is the same for all samples. In
reality, this is not always the case. Therefore, the so-called indirect SFC method that
only measures the signal due to the liquid can be used in order to overcome this
random error on the SFC values. The indirect method is based on a two-step
TD-NMR measurement: First, at the desired measurement temperature, the liquid
part of the NMR signal is measured. Secondly, the sample is tempered at 5  C above
the melting temperature and measured again to obtain the NMR response of the
totally melted sample. The SFC values are calculated based on the two measure-
ments with no need for standard calibration. Additionally, to correct the temperature
effect on the instrument sensitivity, the operator should measure a standard oil known
to be completely liquid at both temperatures. This way, a very precise value of the SFC
can be obtained. Thus, the indirect SFC method is predominant in research and
development (R&D) of new fat compositions although it is not as fast as the direct
method. Regarding the sample preparation, this is done in a similar way as for the
direct method.

Automation System for SFC Analysis

Fully automated SFC analysis systems working unattended are progressively

replacing conventional setups as they provide high throughput (up to 1000 samples
per day, depending on the tempering schemes). The operator using such a setup is
only required for filling the sample in the NMR glass tubes. Loading the samples into
the automation and registering of the samples can be done at any time. The
tempering schemes are precisely followed by the system, while all the results are
stored safely in a database. By precisely following the designed method, automation

TD-NMR in Quality Control: Standard Applications 7

not only ensures reliability but also accuracy and high reproducibility of the mea-
surements. High-tech systems usually combine full robotics with an unprecedented
small footprint. All official SFC methods and more can be implemented with limits
defined by the tempering bath hardware. Automatic and accurate control of temper-
ing times helps in producing crystallization kinetics curves autonomously that can
further be used for structural characterization of the fat.

Conclusion

There is always a need for faster and more reliable analytical equipment for food
producing companies. Pulsed field TD-NMR to measure SFC in the fats and edible
oils is widely used in the food industry. This technique is noninvasive and easy to use
and has excellent repeatability. Due to the multiple advantages offered by NMR
methodology, the popularity of TD-NMR in QC/QA is steadily increasing. For
industries dealing with fat composition, for example, SFC measurement is a power-
ful tool for taking the decision to accept a delivery of raw material at the processing
sites. Moreover, the decision of the fat end product usage (i.e., good for coverage in
chocolate or not) can be facilitated by the measured SFC values. In addition,
TD-NMR instrumentation fits well to QC because of its small footprint, modularity,
and relative low investment and running costs. Automating tempering times and the
whole sample handling ensure highly accurate results in strictest accordance with
SFC’s International Standard Methods. Therefore, a full integration of SFC mea-
surements in food processing production lines will constitute a real-time QC/QA
parameter.

Droplet Size Determination

Introduction

Droplet size distributions play a major role in the development of new products in
nutrition, influencing mouth feel, stability of emulsions, shelf life, microbial keeping
properties, etc. The droplet size distribution characteristics need to be regularly
checked during production quality control, ensuring that the end product will present
the desired characteristics described above. Examples of daily products where
droplet size distributions are verified in both research and development (R&D) and
production are mayonnaise, salad dressing, and soft cheese containing oil droplets
dispersed in a water phase and margarine and low fat spread and butter containing
water droplets dispersed in an oil phase.
Droplet size distribution can be measured by various techniques, TD-NMR,
optical microscopy, electron microscopy, static light scattering, dynamic light scat-
tering, electric pulse counting, etc. The scope of such techniques is summarized in
Table 1.

8 M.F. Cobo et al.

Table 1 Comparison of detection limits of droplet sizing for different techniques

Concentration
Technique Sensitive range (μm) of droplets (%) Sample preparation
Time-Domain or TD NMR 0.5–30 >2 No
Optical microscopy >0.5 0.1–50 Yes
Electron microscopy >0.005 0.1–50 Yes
Static light scattering 0.05–1000 <0.1 Yes
Dynamic light scattering 0.003–5 0.1–30 Yes
Electric pulse counting 0.4–100 <0.1 Yes

In advantage to other techniques, TD-NMR can be used to assess individual

droplet sizes rather than droplet cluster sizes and is equally applicable to free and to
flocculated droplets. Moreover, it requires a minimum sample preparation (fill the
sample in a tube and measure) without dilution so that the samples remain unaltered.
The analyses are straightforward; they do not require specialists and can, hence, be
established easily in production.

d-Var and G-Var Methods

The pulse sequence is based on the stimulated echo with pulsed field gradients
(PFG-STE), where the measurement is performed as function of the gradient (G)
amplitude; see Fig. 2. In this sequence, the stimulated echo is generated by applying
three 90 pulses, the first two being separated by half of the intended stimulated echo
time (τE). Additionally, a pulsed field gradient with amplitude G and duration δ is
applied after the first 90 pulse. The second pulse is used to return the magnetization
parallel to the main magnetic field, where it is kept for typically some few hundreds
of milliseconds (calculated as function of τ E and the gradient pulses separation Δ).
The diffusion process can take place during that time, often referred to a as the
storage time. Afterward the third 90 pulse is applied, followed by a gradient with
the same parameters as the first one, resulting in a stimulated echo at τ E /2 after the
third pulse. The decrease of intensity (S) of the stimulated echo can be related to the
diffusion of the molecules during the storage time.
The experiment is conducted with the so-called G-Var application. Here a series
of gradient experiments are performed under variation of the gradient strength
(G) rather than altering the gradient duration (δ) as in the classical droplet size
measurement δ-Var [11].
The original method (δ-Var) requires the so-called balancing of the pulsed field
gradients as a preparation step. The balancing consumes considerably more time
than the actual measurements, leading to a total time of typically 12–15 min. The
balancing can be understood as a fine adjustment of either the duration or amplitude
of one of the pulsed field gradients (typically the second one) to ensure that the
gradients applied are as close to identical as possible, resulting in a symmetric
stimulated echo at the theoretical temporal position (theoretical echo time based on

TD-NMR in Quality Control: Standard Applications 9

Fig. 2 Pulse sequence used for measuring droplet size distributions: PFG-SE with gradient
amplitude variation (G-Var)

the timings in the pulse sequence). Non-calibrated gradients result in distorted echo
shapes, and the echo top might occur even hundreds of microseconds away from the
theoretical position, leading to an additional change of the signal intensity which will
affect the quantification of the droplet sizes.
The main advantage of the G-Var method is that the balancing procedure is not
needed anymore; thus, the typical analysis time is reduced by a factor of 3, down to a
response time of 5 min or less. This new method, hence, allows for the acquisition of
significantly more data points. Moreover, due to the more stable pulsed field
gradients, the results become more reproducible and the sensitivity is increased.
The detection limit of the target content in the droplet phase is now around 2%,
which corresponds to an increase in target sensitivity by a factor of 5 in comparison
to the original δ-Var method. A detailed description of the G-Var and performance
comparison with δ-Var can be found at [12].

G-Var NMR Requirements

The pulse sequence depicted in the Fig. 2 can be used, with small modifications, for
droplet size measurements, in either oil-in-water (O/W) or water-in-oil (W/O)
emulsions. As this method relies on the measurement of the 1H NMR signal as a
function of gradients, the application requires a relation between the signal S(G) and
the liquid that is confined in the droplet. The 1H nuclei in the continuous phase
should not have contributions to the signal.
For the analysis of oil droplets in water, the weakest gradient (Gmin ) has to be
chosen large enough to suppress the signal coming from the continuous water phase
(free diffusion under gradients). S(G) is then a measure for the signal from the
droplet content only for the full range of gradients used for the experiment.
For the analysis of water droplets in oil, instead of using the free diffusion of the
continuous phase to filter out the signal coming from this component, one can use a
simple T1 filter to guarantee that the signal S(G) is coming from the water inside the
droplet without a contribution from the surrounding oil. For that end, the sequence
illustrated in the Fig. 2 needs a small adaptation, adding a 180 pulse and a delay

10 M.F. Cobo et al.

Fig. 3 G-Var signal acquired as function of the gradient strength (T/m)

before the first 90 pulse. Given the previous knowledge of the T1 relaxation times of
the oil or continuous phase, one can suppress the signal coming from the oil phase by
adjusting the delay between the 180 and the 90 pulses.
In both cases, the adjustments in the measurements mentioned above have to be
executed only once. They remain constant throughout the remainder application.
The adjustments are easily applicable and fast in determination.
Other critical parameters for this sequence are the pulsed gradient duration (δ) and
the pulsed gradient separation (Δ). They are directly linked to the gradient amplitude
(G). It is recommended to choose a combination of settings that allow for the proper
characterization of the decay curve R(%) (normalized amplitudes S(G)/S(G min))
versus G, as exemplified in Fig. 3 for water droplets in oil.

Calibration and Measurement G-Var Concept and Workflow

User-Defined Calibration
Before using G-Var, the system needs to be calibrated. Both the steady gradient and
the gradient amplitude are adjusted to ensure a maximum linewidth corresponding to
a signal decay time of 0.5 ms and an optimal signal intensity. For both cases a doped
water solution 0.5% CuSO 4 5H 2O is typically used. Knowledge of the water self-
diffusion coefficient at the temperature that the calibration is performed is required.
By adjusting the steady gradient, one ensures that the echo top in the experiment is
well defined and that different instruments perform under similar conditions. Such
steady gradient is used later on in all subsequent measurements. The second part of
the calibration correlates the amplitude of the gradient (DC currents) with the gra-
dient strength (T/m). This procedure is fully automatic, requiring a minimal user
input. The recommendation is that the calibration should be performed once every
month.
Using the G-Var application, the calibration curve taken at any temperature in the
range of 5 to 65  C is valid for both oil-in-water and water-in-oil emulsions for the
whole temperature range. This is important as measurements of daily products as

TD-NMR in Quality Control: Standard Applications 11

margarines and butter and measurements of mayonnaise and soft cheese are typically
carried out at 5  C and 20  C, respectively.

Measurement

If the system is configured in a routine operation mode for quality control, no

parameter entry is needed and the measurement can be started right away. Prior to
the start of the experiment, the application automatically defines the receiver gain,
number of scans, and checks if the maximum pulsed field gradient (G end) of the
experiment is suitable for the sample in analysis. In the case that the NMR signal for
G end is below a predefined amplitude, the application automatically readjusts G end
and checks for it again. This way the application makes sure that the curve S(G)
versus G will be well sampled, as exemplified in Fig. 3.
In the data analysis, the first assumption in the current mathematical model used is
that the droplets are spherical, so the droplet size can be estimated by the Murday-
Cotts model [13, 14]:
" #
1
2Δ 2 2 f ðΔ, G, δ, DÞ X
RðΔ, G, δ, D, aÞ ¼ exp   2G γ 2

2 2
 (2)
T2 m¼1 αm α m a  2

where
 2   2 
2δ 2 þ exp α m DðΔ  δÞ  2exp α m Dδ
f ðΔ, G, δ, DÞ ¼ 2  2
αm D

2
 2  αm D  2  (3)
2exp αm DΔ þ exp αm DðΔ þ δÞ
  2
2
α mD

where Δ, G, and δ are the parameters from the pulse sequence described above, D is
the diffusion coefficient of the liquid confined in the droplet, γ is the proton
gyromagnetic constant, α m is the mth positive root of the Bessel function, and a is
the droplet radius.
The expressions above are valid for emulsions which have all droplets with the
same radius a. For food emulsions typically there is a distribution of droplet sizes
(P(a)); in this case the NMR signal decay can be calculated by [15]
Ð1
SðGÞ 0 a3 P ða ÞRðD, a, g, δ Þda
¼ Ð1 3 (4)
SðGmin Þ 0 a P ða Þda

In the current mathematical model, it is assumed that the distribution P(a) is

log-normal, which is typically the case of margarines, butters, salad dressings,
mayonnaises, etc.:

12 M.F. Cobo et al.

Fig. 4 Log-normal distribution of droplet sizes of a butter

( )
2
½ lnð2aÞ  lnð D33Þ
exp 
2σ 2
P ða Þ ¼ p ffiffiffiffiffi (5)
2aσ 2π
where σ is the standard deviation of the logarithm of the droplet diameter and D 33 is
the volume-weighted mean droplet diameter.
Fig. 3 displays the normalized NMR signal (dots) of a commercial margarine
sample and the respective fit (continuous line) accordingly to Eq. 4, from which D 33
and σ are obtained. The corresponding log-normal distribution (Eq. 5) is displayed in
Fig. 4.
As the mathematical procedure above described is performed without user input
and as the data and results are automatically stored, the analysis of sample batches
with automated systems is also possible.

Conclusion

Both D3.3 and σ parameters are important for food researchers to create new food
products with tuned characteristics. Thanks to the new G-Var application, the
determination of droplet sizes in emulsions has not only become faster and more
reliable but is also easier to use. This should allow a further spread of the technique
out of the R&D centers into production for quality control purposes.

Oil and Moisture Content

Introduction

Another classic application of QC TD-NMR in food science and industry is the

oil and moisture content determination. Most commonly the fat content is of
interest. In the past years, an increased interest is noticeable also for the moisture

TD-NMR in Quality Control: Standard Applications 13

content in food products. The extensive experience from previous years shows
that a correct determination of oil and moisture content is possible only for
products with low water content. This is due to the fact that in TD-NMR no
spectral resolution is available and the relaxation properties of oil and water are
too similar so that the commonly known TD-NMR pulse sequences cannot
guarantee a correct separation of signals from these two components. The clas-
sical approach pre-dries the samples in an oven or via infrared or microwave
drying. The drawback of the pre-drying method is that it is labor intensive and
time consuming. Fortunately, new technical developments that were achieved in
the last few years allow the implementation of more advanced experiments that
overcome this issue [16].
This section will focus on TD-NMR methods that are used currently in oil and
moisture determination in food science and industry QC. The first part will review
the so-called traditional method for food with low moisture content, using the spin-
echo technique. The second part will focus on the more advanced method, the
combined relaxation experiment, which can be applied for food with high moisture
content.

Oil and Moisture Content Determination for Low Moisture

Content Food

The application area of TD-NMR has been widened once the Hahn-echo NMR
pulse sequence (Fig. 5) was implemented [17]. In case of food quality control, this
method is used to determine the total oil and moisture content in food samples.
Almost all types of food samples can be analyzed providing that the water content
is minor (typically below 12%); see Table 2. The oil and moisture analyses for
seeds and seed residues have become an International Standard Method [3, 18,
19]. The pulse sequence is presented in Fig. 5. The FID signal amplitude S1 and the
Hahn-echo amplitude S2 are measured. The amplitudes S1 (at τ = 50 μs following
the 90 pulse) and S 2 (at τ = 7 ms) represent the sum of the oil and moisture signals
and the oil signal alone, respectively. Thus, the amplitude difference, S 1–S2, repre-
sents the moisture content. The oil and moisture contents can be measured simulta-
neously after primary calibration. The calibration is performed only once and runs
automatically.
In comparison to other standard methods, TD-NMR has several advantages, for
example, sample preparation kept at a minimum, short analysis time (usually in the
order of seconds), and nonnecessity for chemical solvents. Moreover, TD-NMR
systems are turnkey systems which do not require skilled personnel for operations.
Different sample types (e.g., sunflower, grape, rape seeds) will need different
calibration curves. Generally, three to five samples are sufficient for a calibration.
Reference values obtained by other methods (e.g., Soxhlet extraction or Karl Fischer)
are used to calibrate the TD-NMR equipment.

14 M.F. Cobo et al.

Fig. 5 Hahn-Echo sequence used for oil and moisture determination in food samples

Table 2 Application areas of TD-NMR for the determination of oil and moisture content
Moisture/oil content in all types of seeds and residues
Moisture and fat/oil content in (dry) food:
Cocoa powder, cocoa beans, and chocolate (mass and liquor)
Snacks, cereals, chips, cookies, bread
All types of nuts, beans
Milk powders, bakery powders, baby food, low moisture cheese, soup powders
Flavors, flour, semolina, starch, sugar beet
Moisture and fat content in animal feed

Combined Relaxation Method

The basic understanding of relaxation properties in samples is of major importance

for a proper TD-NMR analysis of food products. It is well known that the transverse
and also the longitudinal relaxation times of water and fat differ. The same is valid
for several other ingredients, for example, proteins. However, as the differences in
relaxation times are relatively small or may overlap, and as magnetization relaxation
depends also on other parameters like temperature, salt concentration, and the mol-
ecule’s local environment, an individual measurement of longitudinal spin-lattice or
transversal spin-spin relaxation times (T 1 or T2 ) is not sufficient for getting a good
correlation with sample properties. Therefore, a combined relaxation experiment has
been developed, where the magnetization at a given time is determined by both the
longitudinal and the transversal relaxation. There are several approaches to realize
this aim. One of them uses a steady-state dynamic equilibrium [20]; others make use
of the longitudinal recovery of magnetization and measure the transversal relaxation
at several points of the T1 curve by CPMG (named after Carr, Purcell, Meiboom, and
Gill [21, 22]). Using a modified sequence which also acquires the FID in addition to
the echoes, solid components can also be measured and analyzed. Thus, the relax-
ation properties T 1(M(t, T 2), M(t, T 2 *)), T2 (M(t, T1 )), and T 2*(M(t, T1)) can be
measured within one experiment. The information which can be obtained by this
experiment is huge and not easy to be analyzed quantitatively in terms of classic
relaxation time analysis. Therefore, another approach was chosen for data pro-
cessing, using statistical, i.e., chemometric methods [23], which are common, for

TD-NMR in Quality Control: Standard Applications 15

example, in near-infrared studies. A correlation with several sample properties can

thus be established.

Results

The combined relaxation analysis is more and more applied in different food
industries with potential to become a QC method in the future. First results were
presented in [24] by Guthausen et al. The authors applied this new pulse sequence,
which is presented in more detail in [25], on samples like margarines, mayonnaises,
and sauces. Depending on the typical sample size, different system configurations
can be selected. Data processing is performed using chemometric methods, allo-
wing several possibilities of data preprocessing, after which the correlation is
established based on reference values by applying the PLS (partial least square)
algorithm.
Classic simple T 1 or T2 relaxation measurements proved to be not sufficient to
establish a correlation with extraction values. In [26], transversal magnetization
decays of food are analyzed showing the difficulties in the analytical approach of
fitting and interpreting multi-exponential decays. The authors use the chemometric
approach to analyze the data. Taking into account the complexity of the materials, it
was not surprising that the approach of using discrete decay functions was not
satisfactory. Therefore, in the mentioned study, only the chemometric approach
was used for data analysis. Different food samples like mayonnaise and margarine
were examined measuring the longitudinal, the transverse, and also the combined
magnetization decays. The correlation parameters obtained from the chemometric
data analysis demonstrated that the best correlation with the reference fat content for
the samples under investigation was achieved by applying the combined relaxation
time analysis. It is obvious that the correlation was improved by combining the
information of T 1 and of T2 experiments.
As it is well known that relaxation times reflect dynamic and to some extent also
static properties of the surrounding of the 1 H nuclei, it is obvious that already small
changes in the sample composition change the relaxation to an extent which
doesn’t correlate with the concentration of a certain component. The combined
relaxation method contains both relaxation processes, therefore increasing the
probability of being sensitive to the wanted quantity, fat, moisture, or other
components. The fat content as determined by the combined relaxation method
in [24] was correlated with the values obtained by the reference method. The
correlation could be established over a wide range of fat content as well as for
different food products. The correlation coefficient amounts to 0.994, the F-factor
to 2814. The correlation considered 35 different samples with a fat content
between 9.2% and 82.1%.
Another study in which the combined relaxation method was applied successfully
was reported by Castell-Palou et al. [27], where the authors analyzed fat and water
content in cheese samples. A total number of 40 cheese samples from different

16 M.F. Cobo et al.

Spanish regions, of different ripening stages, were measured and compared to their
reference water and fat content. The fat content varied from 26% to 46% and the
water content varied from 10% to 45%. The coefficients of determination were
higher than 0.97 for both fat and water contents.
Horn et al. [28] used the combined relaxation method to quantify oil and protein
in cotton seeds by TD-NMR. Varying amounts of delinted cottonseeds were used
for 42 different genotypes of the calibration seed to cover a wide range of protein
and oil values. The authors noticed that in their experiments, all three components
of the modified combined relaxation sequence (FID, solid echo, and CPMG) were
important for accurate protein prediction. A very strong prediction was found for
the oil content, with a R 2 of 0.998. For the protein correlation with reference
(nitrogen) protein values obtained with the Dumas method, a R 2 of 0.95 was
obtained.

Conclusion

Two main methods were presented for oil and moisture content determination by
TD-NMR, the traditional spin-echo technique, and the combined relaxation analysis.
These methods are fast and accurate as well as all the other TD-NMR methods,
providing a tool for the analysis and quality control of food products.

Conclusions

In this chapter we presented some of the standard TD-NMR applications and their
variants and the employment of automation to make these applications more suitable
for industrial use. Combining automation with TD-NMR-based technology and its
applications increases the power of in-process control (IPC) during production. It
allows better quality and process control by higher sample throughput in manu-
facturing which results in reduced human error and operation cost. It also increases
the possibility to implement this technology in processing and product manufactur-
ing for 100% IPC.
Although TD-NMR-based applications are not as widely used as other technol-
ogies such as near-infrared-based applications, TD-NMR is breaking limits where
other analytical techniques cannot be used and it can provide complimentary support
or even a possible replacement for other methods to be used in food quality control.
TD-NMR-based applications are evolving over time as a result of an increased
awareness and an active collaboration among analytical instrument and application
providers, food industries, and food scientist around the globe.
TD-NMR-based solutions and methods are highly received and acknowledged by
official regulatory bodies such as AOCS/ISO/ASTM/IUPAC and have been adopted
by several food companies. We hope to bring forth these methods and future pos-
sibilities to the attention of the reader.

TD-NMR in Quality Control: Standard Applications 17

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Citations (0) References (25)

Modified spin-echo method for measuring nuclear relaxation times

Article Jan 1966 · REV SCI INSTRUM

S. Meiboom · D. Gill

View

Spin Echoes

Article Jan 1950 · Phys Rev

E.L. Hahn

View Show abstract

Simultaneous Quantification of Fat and Water Content in Cheese by TD-NMR

Article Oct 2013 · FOOD BIOPROCESS TECH

África Castell-Palou · Carmen Rosselló · Antoni Femenia · Susana Simal

View Show abstract

Measurement of Oil Droplet Size Distributions in Food Oil/Water Emulsions by Time Domain Pulsed Field Gradient NMR

Article Jul 2001 · J COLLOID INTERF SCI

G.J. W. Goudappel · John P M van Duynhoven · M.M.W. Mooren

View Show abstract

Pulsed NMR Studies of Restricted Diffusion. I. Droplet Size Distributions in Emulsions

Article Aug 1972 · J COLLOID INTERF SCI

Kenneth J. Packer · C. Rees

View Show abstract

Effect of Diffusion on Free Precession in Nuclear Magnetic Resonance Experiments

Article May 1954 · Phys Rev

HY CARR · EM PURCELL

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Self‐Diffusion Coefficient of Liquid Lithium

Article Jun 1968 · J CHEM PHYS

James S Murday · R. M. Cotts

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Scope of droplet size measurements in food emulsions by pulsed field gradient NMR at low field

Article Dec 2002 · Magn Reson Chem

John P M van Duynhoven · G.J. W. Goudappel · Gerard VAN Dalen · A. P. A. M. Eijkelenboom

View Show abstract

Water/moisture and fat analysis by time-domain NMR

Article Jun 2006 · FOOD CHEM

Harald Todt · Gisela Guthausen · Wolfgang Burk · Andreas Kamlowski

View Show abstract

A rapid benchtop NMR method for determination of droplet size distributions in food emulsions

Article Nov 2007 · Eur J Lipid Sci Tech

John P M van Duynhoven · Benjamin Maillet · Jimmy Schell · Dagmar Van Dusschoten

View Show abstract

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