NMR Spectroscopy in Food Analysis

Apostolos Spyros, Photis Dais

• Publisher: Royal Society of Chemistry
• Year: 2015
• ISBN:
  9781782625667
• Edition: 1
• Topic: Technology & Engineering
• Subtopic: Food Science
• Index: Technology & Engineering

During the last two decades, the use of NMR spectroscopy for the characterization and analysis of food materials has flourished, and this trend continues to increase today. Currently, there exists no book that fulfils specifically the needs of food scientists that are interested in adding or expanding the use of NMR spectroscopy in their arsenal of food analysis techniques. Current books and monographs are rather addressed to experienced researchers in food analysis providing new information in the field. This book, written by acknowledged experts in the field, fills the gap by offering a day to day NMR guide for the food scientist, affording not only the basic theoretical aspects of NMR spectroscopy, but also practical information on sample preparation, experimental conditions and data analysis. Current developments in the field covered in this book are the availability of solid state NMR experiments such as CP/MAS and more importantly HR-MAS NMR for the analysis of semisolid foods, and the increasing use of chemometrics to analyze NMR data in food metabonomics. Moreover, this book contains an up to date discussion of MRI in food analysis including topics such as food processing and natural changes in food such as ripening. The book is a compact and complete source of information for food scientists who wish to apply methodologies based on NMR spectroscopy in food analysis. It contains information so far scattered in the primary literature, in NMR treatises and food analysis books, in a concise format that makes it appealing to food scientists who have no or minimal experience in magnetic resonance techniques. The inclusion of practical information about NMR instrumentation, experiment setup, acquisition and spectral analysis for the study of different food categories make this book a hands-on manual for food scientists wishing to implement novel NMR spectroscopy-based analytical techniques in their field.

CHAPTER 1

Introduction

Nuclear magnetic resonance (NMR) spectroscopy is an effective analytical technique, which has been used systematically in food analysis and authentication in recent years. Its origin is traced back to 1946 when two groups of scientists at Harvard University (Purcell, Torrey and Pound) and at Standford University (Bloch, Hansen, and Packard), working independently, observed proton resonance signals from paraffin wax and water, respectively. For their discovery, Purcell and Bloch were jointly awarded the Nobel Prize in Physics in 1952.

The first application of NMR in food science dates back to 1957 when low-resolution NMR measured moisture in foods. Consistent and widespread application of NMR in food science started in the 1980’s mainly due to deficiencies in instrumentation and the complexity of food matrices. Since then, an explosive publication of research and review articles dealing with NMR applications in food science has appeared in scientific journals and several books. Figure 1.1 shows diagrammatically the explosion of publications in food science after 1988.

Figure 1.1 The graph presents the number of publications per year that include the words ‘food’ and ‘NMR’ or ‘MRI’(either as acronyms or in full text) in their title, keywords or abstract, as indexed in Scopus.
(Retrieved on May 25, 2012 from www.scopus.com.)

Also, numerous oral and written communications have been presented in domestic and international conferences. In particular, an International Conference on Application of Magnetic Resonance in Food Science is held in Europe every two years. This conference started in 1992 and gave the opportunity for scientists worldwide to present new applications of NMR in food science and technology. It is worth mentioning that NMR methods have been approved as official methods by the European Union (e.g. detection of wine fraud). There are several reasons for this development: (a) the increasing sophistication and the user-friendly NMR instrumentation; (b) the increasing need of the food industry to understand innovate its products and processes; and (c) the necessity for the development of new and more effective analytical techniques for the quality control and authentication of foods and thereby the reinforcement of pertinent legislation.

Foods are very complex and highly heterogeneous systems comprising a large number of chemical compounds, the composition of which varies considerably under certain circumstances (e.g. agronomical or slaughter practices, industrial processes, storage, maturation, etc.). To this direction, one-dimensional (1D) liquid or solid-state high-resolution NMR spectroscopy can provide in a single experiment a wealth of structural and quantitative information in the form of the NMR parameters, namely chemical shifts, coupling constants and signal intensities. For the same sample the researcher can choose different nuclei, such as¹H,¹³C,³¹P,¹⁹F—to mention the most popular nuclei—that allow the study of food samples under different perspectives and to extract the maximum information about its natural or industrial condition. These experiments need no separation of the various food components and no serious sample pre-treatment. Moreover, NMR spectroscopy is sensitive to dynamics, which allows differentiation between molecules or groups of molecules with different mobility through spin–lattice and/or spin–spin relaxation measurements.

In cases where the complexity of the food sample is so severe, causing extensive signal overlap in 1D spectra, the arsenal of NMR spectroscopy provides a large number of analytical techniques starting from the homonuclear and heteronuclear multi-dimensional NMR to its hyphenation with effective separation techniques, such as liquid chromatography (LC-NMR). In particular, two-dimensional (2D) NMR techniques, such as COSY, TOCSY, NOESY, HSQC, etc., based on the inherent ‘communication’ of nuclei with each other (through spin–spin and dipolar coupling), spread out the spectroscopic information in two dimensions unravelling hidden nuclear connectivities and facilitating the structural characterisation of the molecules in the food sample. Although NMR spectroscopy is not a destructive analytical technique and recovery of the analyte can be easily achieved after experimentation, industrial needs may require the examination of food products under different processing conditions by non-invasive means. Magnetic resonance imaging (MRI), used extensively in medicine, has been exploited in recent years in food analysis. The ability of MRI to show spatial resolution within the food product and the judicial application of MRI techniques allows the monitoring of the fate of certain molecules (e.g. water) and reveals various molecular interactions and changes in tissue structure that occur during food processing or storage (e.g. food freezing and thawing).

The combination of NMR spectroscopy with multivariate statistical methods provided an alternative possibility of analysing and maximising the information recovery from complex NMR spectral data of foods. This methodology, usually called metabonomics, does not necessarily require the identification of the individual signals in the spectrum as in quantitative NMR, but seeks to find subtle spectral features that can identify unequivocally the presence of metabolites or useful biomarkers. Pattern recognition techniques (supervised or unsupervised) can be used to map the NMR spectra of a large number of samples, and locate spectral fingerprints that reflect either metabolic changes or used to distinguish sample classes.

The disadvantage of the early days of NMR spectroscopy related to the low sensitivity and high cost of the analysis does not hold true for the NMR instrumentation of the present day. These drawbacks have been largely compensated by the development of modern hardware comprising strong magnetic fields up to 23.5T and cryogenic probes that allow easy detection of food components at the level of μg and even ng. Moreover, the progress in sophisticated software and innovations in automation allow the screening of a large number of samples (overnight run), reducing the experimental time to a few minutes even for the less sensitive nuclei (e.g.¹³C,¹⁵N).

In concluding this introductory chapter, we could add that it is not only the unique information that NMR provides, but also the versatility of methods, instruments and probes that make it an important tool for qualitative and quantitative analysis.

This book has been organised as follows: Chapter 1 is the book’s introduction. Chapter 2 gives an account of the theory underlying the physical phenomenon of NMR and grouping the most useful NMR techniques to better understand the core principles, which appear in subsequent chapters. Since NMR is a well-documented spectroscopic technique and it is well described in several introductory and advanced books, this chapter will be kept to a minimum. Chapter 3 describes the NMR instrumentation in an attempt to familiarise the reader with the hardware and software components of modern high-resolution and solid-state NMR spectrometers, and their functions and automation. Relevant information about NMR spectrometers and the implementation of its components may help the reader to choose the right spectrometer and accessories for their needs. Also, this chapter includes useful information about the hardware systems and experimental designs to perform sophisticated experiments, such as (HP)LC-NMR, time-domain NMR, and high-throughput and on line NMR. Appropriate guidance for obtaining pure samples from various food matrices that are suitable for NMR experiments will be presented in Chapter 4, whereas the experimental conditions described in Chapter 5 may help the NMR user to choose the right input values for the critical parameters in the experimental setup in order to obtain the maximum possible information from the NMR experiment, and to perform quantitative analyses with high accuracy and precision. Chapter 6 presents a few aspects of the supervised and unsupervised pattern recognition statistical methods employed for data exploration, classification of food samples, and the build-up of calibration–prediction models giving special attention to NMR metabonomics. The applications of NMR spectroscopy and its specialties to different food systems are discussed in Chapters 7–11. A detailed presentation of the available NMR methodologies and techniques for each food category is provided, whereas practical guidance and tips for performing concrete experiments is afforded. Every chapter starts with a short abstract and ends with relevant bibliographic coverage.

CHAPTER 2

Theoretical Aspects

2.1 Nuclear Spins and Energy States

An atomic nucleus is a collection of protons and neutrons (nucleons) that possess a quantum mechanical property called spin, which is characterised by spin angular momentum. Spin angular momentum is an intrinsically quantum mechanical property that does not have a classical analog. All subatomic particles are spin

particles. The nucleus itself has a total spin angular momentum formed by the coupling of the individual spin angular momenta of its constituent protons and neutrons. The total nuclear spin angular momentum quantum number I may therefore take values: 0, 1/2, 1, 3/2, 3, 5/2, etc. A nucleus with non-zero quantum numbers I behaves as a small magnet or magnetic dipole with a magnetic moment μ. The magnetic moment is an intrinsic property of the nucleus, and it is associated with the angular momentum of the nucleus. As a vector, nuclear magnetic moment has two properties: magnitude and direction. The magnitude of μ is quantised and given by eqn (2.1):

(2.1)

where

is Plank’s constant and γ is the magnetogyric ratio, an inherent property of the nucleus. This parameter is unique for each nucleus. When I=0, then μ is zero, and the nucleus does not have magnetic properties. Nuclei with μ=0, such as¹²C,¹⁶O,³²S cannot be studied by NMR. The smallest magnetic moments belong to protons and is called the nuclear magneton, μ_N; its value is calculated from

(e is the electric charge, m_p is the mass of proton, and c is the velocity of light) to 5.0505×10⁻²⁷ J T⁻¹. Under the influence of an external magnetic field of intensity (strength)B₀ fixed along the z-axis of a static Cartesian coordinate system, the magnetic moment assumes discrete orientations (the nuclear Zeeman effect) governed by the magnetic quantum number m_I. The allowed values of m_I are −I, −I+1, −I+2, …, I−1, I, giving rise to 2I+1 possible orientations. Each orientation defines an energy level or state, with energy:

(2.2)

with m_I=+

or−

for nuclei with I=

.

is the projection of the magnetic moment along the z-axis. The orientation of μ_z with respect to B₀ defines the nuclear energy states. For I=

, μ_z parallel to B₀ defines the energy state with the lower energy, whereas its anti-parallel orientation identifies the energy state with the higher energy. It is traditional to label the low and high-energy states with the Greek letters α and β, respectively. The α state with m_I=+

is often described as ‘spin up’, and the β state with m_I=−

as ‘spin down’(Figure 2.1).

Figure 2.1 The orientation of the magnetic moment of a nuclear spin with I=

. Each orientation defines an energy state. The energy gap ΔE depends on the static magnetic field strength B₀ and the gyromagnetic ratio of the nucleus.

The energy space (eqn (2.3)) between the two states is:

(2.3)

where, E_β ...