Table Of ContentApplications of NMR Spectroscopy
Applications of NMR Spectroscopy
(Volume 1)
Edited By
Atta-ur-Rahman, FRS
Kings College
University of Cambridge
Cambridge
UK
&
M. Iqbal Choudhary
H.E.J. Research Institute of Chemistry
International Center for Chemical and Biological Sciences
University of Karachi
Karachi
Pakistan
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ISBN: 978-1-60805-963-8
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PREFACE
Nuclear Magnetic Resonance (NMR) spectroscopy is one of the most powerful and robust
techniques for the study of structures. NMR spectroscopy has been extensively applied in various
fields, such as structural chemistry, structural biology, medicinal chemistry, food and
environmental analyses, forensic sciences, and biomedical and diagnostic sciences. Most of the
available books and monographs on NMR spectroscopy cover only a specific topic and it is rare to
find a book series which provides in-depth state of the art reviews of applications of NMR
spectroscopy in diverse fields. “Applications of NMR Spectroscopy” is an attempt to fulfil this
need. The first volume of the series is an excellent compilation of very well written reviews,
contributed by several leading practitioners of NMR spectroscopy.
The first two reviews deal with the analysis of fats and oils by NMR spectroscopy. Physical and
chemical properties, palatability, and shelf life of prepared food, meat and dairy products, and
other edibles largely depend on their lipid components. Zhang et al have reviewed the use of Low
Field Pulsed NMR Spectroscopy (LFP-NMR) in determining the liquid-solid ratio in various lipids
compositions. Solid-liquid ratio is an important parameter which defines various physico-chemical
properties of lipids, including particle size distribution in emulsions, fat crystallization, and quality
control during food storage and transportation. Interestingly low or ultra-low pulse NMR
spectroscopy is increasingly used for a variety of analyses due to its robustness and the availability
of cost effective instrumentation.
NMR methods have been proven to be robust, reproducible, and accurate, apart from being non-
invasive and non-destructive, for the analysis of edible oils and fats. Sherazi and Mahesar have
contributed a chapter on the use of NMR spectroscopic methods for the analysis of the quality of
edible oils.
Emwas et al have presented an excellent review on the potential applications of NMR based
metabolomics and metabonomics approaches for the diagnosis of human diseases, such as cancers,
cardiovascular and metabolic disorders and neurological diseases. NMR-based metabolomics has
several advantages over other techniques, such as identification of new and novel biomarkers in
disease conditions, as well as its compatibility with the diverse nature of biological fluids on which
metabolomics is conducted. The authors have provided an in depth commentary of various factors
that influence the metabolic balance, which can affect the outcomes of the NMR-based
metabolomic study.
The review by Siniscalco and Antonucci in concerned with the use of NMR based diagnosis of
autism and related disorders. Autism spectrum disorders (ASDs) are now regarded as a global
health challenge. Our understanding of the ASDs at the molecular level is still very limited due to
their complex and heterogeneous nature. Early diagnosis has special merit in controlling the
prevalence of ASDs. Proton magnetic resonance spectroscopy (MRS) is a non-invasive technique
for the study of chemical and cellular changes that occur during the on-set and progression of
ASDs. Early signs of abnormalities at biochemical and cellular levels, related to ASDs, can be
successfully monitored by MRS methods.
Rieko Ishima has contributed a very well written and thoroughly referenced review on the use of
NMR spectroscopy in the study of protein-inhibitor interactions. The understanding of receptor-
viii Preface
ligand interactions is of crucial significance in lead discovery and optimization in the drug
discovery process. Ishima has provided an in depth description of various NMR techniques
including STD (Saturation Transfer Difference) methods used in the study of protein-inhibitor
interactions at molecular and atomic levels.
The identification of chiral molecules is a major challenge in structural chemistry, particularly
because of the regulatory requirement of pharmaceutical products of high enantiomeric purity that
demands such methods. Uccello-Barretta et al have discussed the important applications of NMR
spectroscopy in the identification and quantification of chiral drugs and their metabolites. NMR
spectroscopy offers several methods which can meet such demands in drug discovery and
development as well as in drug quality monitoring.
We are very much indebted to the contributors of the various reviews who deserve our special
appreciation for their hard work and intellectual inputs in this important field. We would like to
thank the entire team of Bentham Science Publishers, especially Mr. Mahmood Alam (Director
Bentham Science Publishers), and Ms. Fariya Zulfiqar (Assistant Manager Publications) for the
efficient management of the first issue of the book series.
Atta-ur-Rahman, FRS
Kings College
University of Cambridge
Cambridge
UK
&
M. Iqbal Choudhary
H.E.J. Research Institute of Chemistry
International Center for Chemical and Biological Sciences
University of Karachi
Karachi
Pakistan
List of Contributors
Abdul-Hamid M. Emwas
NMR Core lab, Biological and Environmental Sciences and Engineering, King
Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia
Dario Siniscalco
Department of Experimental Medicine, Division of Pharmacology, Second
University of Naples, Italy; Centre for Autism – La Forza del Silenzio, Caserta,
Italy and Cancellautismo – Florence, Italy
Federica Aiello
Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via
Risorgimento 35, 56126 Pisa, Italy
Federica Balzano
Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via
Risorgimento 35, 56126 Pisa, Italy
Gloria Uccello-Barretta
Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via
Risorgimento 35, 56126 Pisa, Italy
Hacene Serrai
King Abdullah International Medical Research Center, Jeddah, Kingdom of Saudi
Arabia and Department of Electrical Engineering, University of Manitoba,
Winnipeg, Canada
Hong Zhang
Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,
Pudong, Shanghai, P. R. China
Jasmeen S. Merzaban
NMR Core lab, Biological and Environmental Sciences and Engineering, King
Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi
x List of Contributors
Arabia and King Abdullah International Medical Research Center, Jeddah,
Kingdom of Saudi Arabia
Lu Zhang
Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,
Pudong, Shanghai, P. R. China
Nicola Antonucci
Biomedical Centre for Autism Research and Treatment, Bari, Italy
Rieko Ishima
Department of Structural Biology, University of Pittsburgh, School of Medicine,
Pittsburgh, PA 15260, USA
Roberta Settambolo
CNR-ICCOM, UOS di Pisa, Via Risorgimento 35, 56126 Pisa, Italy
Sarfaraz Ahmed Mahesar
National Centre of Excellence in Analytical Chemistry, University of Sindh,
Jamshoro, 76080-Pakistan
Shichao Xie
Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,
Pudong, Shanghai, P. R. China
Syed Tufail Hussain Sherazi
National Centre of Excellence in Analytical Chemistry, University of Sindh,
Jamshoro, 76080-Pakistan
Xiaoyang Sun
Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,
Pudong, Shanghai, P. R. China
Applications of NMR Spectroscopy, Vol. 1, 2015, 3-56 3
CHAPTER 1
Applications of Low-Field Pulsed Nuclear Magnetic Resonance
Technique in Lipid and Food
Hong Zhang*, Lu Zhang, Xiaoyang Sun and Shichao Xie
Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,
Pudong, Shanghai, P. R. China
Abstract: The ratio between liquid and solid portion of lipid can be determined quickly
and accurately by low-field pulsed nuclear magnetic resonance (LFP-NMR) instrument.
This analytical tool has become one of important techniques to characterize product
physical properties especially related to fat melting behavior, mouth feeling, and
cooling effect etc. in lipid and food application system. In lipid, it can be used to
determine the solid fat content, evaluate the crystallization rate and the compatibility of
lipid blends, monitor the enzymatic interesterification degree, and estimate the fat
crystal type. As a nondestructive testing, LFP-NMR technique was also applied to
analyze the particle size distribution of emulsion, the fat crystallization, and the quality
control during food storage. LFP-NMR is not only applied to evaluate the fat
crystallization, but also to analyze the crystallinity of sugar. These application
progresses of LFP-NMR technique in lipid and food will be summarized in this chapter.
Keywords: Compatibility, crystallinity, crystallization rate, emulsion, foods,
interesterification, lipids, low-filed pulsed NMR, particle size distribution,
polymorphism, SFC.
1. INTRODUCTION
Low-field pulsed nuclear magnetic resonance (LFP-NMR) is now a standard
analytical technique which has been successfully applied in the study of lipid and
food. LFP-NMR also known as time-domain NMR (TD-NMR) started about 40
years ago in the cooperation between Unilever Research (The Netherlands) and
Bruker Physik AG. The idea was raised to build a small tabletop TD-NMR
analyzer for the solid-to-liquid ratio analysis on fat compositions [1]. Nowadays,
its applications do not only cover research and development but also quality and
process control in the food supply chain. It has been used throughout all areas of
*Corresponding author Hong Zhang: Wilmar (Shanghai) Biotechnology Research & Development Center Co.,
Ltd. Shanghai, P. R. China; Tel: +86-21-31153106; Fax: +86-21-58487667;
E-mail: [email protected]
Atta-ur-Rahman and M. Iqbal Choudhary (Eds)
Copyright © 2015 Bentham Science Publishers Ltd. Published by Elsevier Inc. All rights reserved.
10.1016/B978-1-60805-963-8.50001-9
4 Applications of NMR Spectroscopy, Vol. 1 Zhang et al.
food science and technology to characterize product physical properties especially
related to fat melting behavior, mouth feeling, and cooling effect etc. [2-6].
LFP-NMR is a fast and accurate alternative method compared to the destructive
conventional chemical methods to determine the content of water and fat
simultaneously. The analysis is non-destructive, requires less space and does not
require inflammable chemicals and expensive glassware. It is not only a standard
technique for the determination of solid fat content (SFC), but also a well-established
technique for routine analysis, e.g., determination of oil content of seeds, meals and
meat, and determination of humidity in a variety of foodstuffs [7].
In 1960s, Conway first used NMR to analyze whole seed for oil content. Although
wide-line NMR has been in use since 1980s for selecting seeds of higher oil
content in plant-breeding programme, LFP-NMR provides a faster and more
accurate approach for the determination of oil in seeds. Without weighing and
oven drying the seeds, NMR takes about 10 sec per analysis. Seeds can
subsequently be used in breeding programme [8].
A perfect process analytical method would be based on a robust, non-invasive and
easy to handle customized technique operating in real-time. The ideal
instrumentation comes without any need for calibration, is an absolute method,
has a professional support, and is compliant to increasing regulatory requirements.
A trend can be observed towards such an all-in-one device suitable for every
purpose in NMR. Almost all types of food samples could be analyzed in the case
the water content is minor (typically below 15%) [9]. However, samples with the
high amounts of water/free water cannot be treated this way. The NMR signal
from water will interfere with the oil signal. This is because the relaxation times
of free water and oil are rather similar and chemical shift dispersion is obscured in
LFP-NMR by the magnets inhomogeneity. Moreover, the selectivity in commonly
used LFP-NMR pulse sequences is too small to guarantee a clear separation of
signals. The classic approach was to pre-dry the samples either for example by an
oven, chemical reagents like CuSO4 or via infrared or microwave drying
processes, rendering the application a two-step approach. Under these
circumstances, samples (like olives, sausages, fish, and meat) need to be pre-dried
to remove free water.
Applications of Low-Field Pulsed NMR Applications of NMR Spectroscopy, Vol. 1 5
Most common LFP-NMR applications are based on very simple NMR pulse
sequences, like the free induction decay (FID) or the Hahn-echo acquisition. A
Hahn-echo sequence is adapted to determine the total oil and moisture content in
food samples simultaneously by using relaxation properties as contrast parameter
[2]. The analyses of oil and moisture for seeds and seed residues have become
International Standards Methods [10, 11]. According to these methods, both the
FID signal amplitude S , and the echo amplitude S , are measured (Fig. 1). The
1 2
amplitudes S (at about 50 µs following the 90° pulse) and S (at τ = 7 ms)
1 2
represent the sum of the oil and moisture signals and oil content, respectively.
Thus, the amplitude difference S -S , is related to the moisture content. However,
1 2
for samples with high amounts of free water (like olives, sausages, fish, and meat)
samples need to be pre-dried to remove free water since the relaxation times of
free water and oil are rather similar. This method has found more than a hundred
of users only in Spain for the precise oil content determination in olive paste [12].
LFP-NMR method can also be used to measure fat content in chocolate mass and
paste as well as in chocolate liquor.
LFP-NMR may be used to analyze most food (i.e., fish, meat, dairy) products along
the production process from the raw material until the finished product. Examples of
applications in food systems are Rheo-NMR, i.e., performing rheological profiling
of complex fluids [13, 14] and NMR-baking, i.e., monitoring the changes in the
states of water during the dough-to-bread baking process [15]. The transitions in the
states of water occurring at different temperatures were studied in the process of
cooking meat by cooking inside the NMR magnet [16]. It was found that a new
water population was developed in meat as a result of cooking.
(cid:2)(cid:4)(cid:3) (cid:2)
(cid:2) (cid:2)
(cid:2) (cid:4)
(cid:2) (cid:2)
(cid:3) (cid:4)
(cid:2)(cid:3)(cid:4)(cid:5)
Figure 1: Hahn-echo NMR pulse sequence with amplitudes and amplitude difference for the
determination of both the moisture and fat content of low water products [2].
