Otles methods of analysis of food components and additives

Methods of
Analysis of Food
Components
and Additives


Chemical and Functional Properties of Food Components Series
SERIES EDITOR

Zdzislaw E. Sikorski
Chemical and Functional Properties of Food Proteins
Edited by Zdzislaw E. Sikorski

Chemical and Functional Properties of Food Components, Second Edition
Edited by Zdzislaw E. Sikorski

Chemical and Functional Properties of Food Lipids
Edited by Zdzislaw E. Sikorski and Anna Kolakowska

Chemical and Functional Properties of Food Saccharides
Edited by Piotr Tomasik

Toxins in Food

Edited by Waldemar M. Dabrowski and Zdzislaw E. Sikorski

Methods of Analysis of Food Components and Additives
Edited by Semih Ötles,

Methods of
Analysis of Food
Components
and Additives
EDITED BY

Semih Ötles,
Ege University
Department of Food Engineering
Izmir, Turkey

Boca Raton London New York Singapore

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Taylor & Francis Group, the academic division of T&F Informa plc.


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Preface
The ability to accurately separate, identify, and analyze nutrients, additives, and
toxicological compounds found in food and food products has become critically
important in recent decades, as knowledge of and interest in the relationships

between diet and health have increased. This requires training students and analysts
in the proper application of the best methods, as well as improving, developing, or
adapting existing methods to meet specific analytic needs. This book aids the analyst
by providing a valuable reference to both newly developed and established methods
of analysis of food components and additives.
The book comprises 16 chapters, which take the reader through brief and accessible descriptions of methods of analysis of food components and additives. Ranging
from chemical analysis of food components and additives to infrared (IR), nuclear
magnetic resonance (NMR), Fourier transform Raman (FTR), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), and more. The book provides first-hand explanations of modern methods, contributed by 24 leading scientists, many of whom
actually developed or refined the techniques, and presents new documented information on standard methods of analysis of food components and additives in a uniform
format and in a style that can be understood by a reader who is not familiar with the
analysis of each component. Each chapter is structured to provide a description of
the information about the component or additive that can be analyzed, a simple
method explanation of how it works, examples of applications, and references for
more detailed information. This format also facilitates comparison of methods of
analysis of each component. The use of different authors to cover a broad spectrum
of methods resulted in some differences of style, but overall the book achieves its goal
The first chapter, “Selection of Techniques Used in Food Analysis,” covers topics
relevant to all techniques, including sample preparation, quantitative measurements,
and information management, and concentrates on what goals can be achieved by
applying different techniques for various purposes in food analysis. The second
chapter, “Statistical Assessment of Results of Food Analysis,” provides an overview
of the need for statistical assessment of the results of food analysis and the evaluation
of most suitable methods for different situations at a level that is more complete
than those found in most introductory analysis textbooks. The remaining 14 chapters
address the major areas of analysis of food components and additives: analysis of
drinking waters, proteins, peptides, amino acids, carbohydrates, food lipids, metals
and trace elements in foods, vitamins, carotenoids, chlorophylls, food polyphenols,
aroma compounds, food volatiles, sensory analysis of foods and determination of
food allergens, genetically modified components, pesticide residues, pollutants in
foods, chemical preservatives in foods, radioactive contaminants in foods, and rapid

analysis techniques in food microbiology. In most chapters, many examples of


applications of methods to analytical problems are provided. The references provided
in these chapters can be highly useful and valuable for those seeking additional
information.
This comprehensive book should serve as a reference for scientists, analytical
chemists, engineers, researchers, food manufacturers, personnel from government
agencies, standards writing bodies, students majoring in various science disciplines
(biology, biochemistry, chemistry, environmental science, engineering, and food
chemistry, to name a few) interested in obtaining a stronger background in analysis,
and all those involved in the analysis of both food components and food additives.


The Editor
A native of Izmir, Turkey, Semih Ötles¸ obtained a B.Sc. degree from the Department
of Food Engineering (Ege University) in 1980. During his assistantship at Ege
University in 1985, he received an M.S. in food chemistry, and in 1989, after
completing his thesis research on the instrumental analysis and chemistry of vitamins
in foods, he earned a Ph.D. in food chemistry from Ege University. In 1991–92, he
completed postdoctoral training, including an OECD postdoctoral fellowship, at the
Research Center Melle at Ghent University, Belgium. Afterward, Dr. Ötles¸ joined
the Department of Food Engineering at Ege University as a scientist of food chemistry, being promoted to associate professor in 1993 and to professor in 2000. During
1996–1998 he was deputy director at the Ege Vocational School of Higher Studies.
Since 2003 he has been vice dean of the engineering faculty, Ege University.
The research activities of Professor Ötles¸ have been focused on instrumental
analysis of food compounds: he began a series of projects on the separation and
analysis techniques of high-performance liquid chromatography (HPLC), first for
analysis of vitamins in foods, then proteins and carbohydrates, and, most recently,
carotenoids. Other activities span the fields of GC, GC/MS analysis, soy chemistry,

aromatics, medical and functional foods and nutraceutical chemistry; included are
multiresidue analysis of various foods, and n-3 fatty acids in fish oils.
Professor Ötles¸ is the author or coauthor of more than 150 publications (technical papers, book chapters, and books) and a presenter of seminars. He is a member
of several scientific societies, associations, and organizations, including the Asian
Pacific Organization for Cancer Prevention (APOCP) and the International Society
of Food Physicists (ISFP). He is a member of the steering committee of APOCP’s
local scientific bureau and is the Turkish representative of ISFP, and has organized
international congresses on diet/cancer and food physics.
He is a member of editorial advisory boards for Asian Pacific Journal of Cancer
Prevention; Food Science & Technology Abstracts of IFIS (International Food Information Service); Current Topics in Nutraceutical Research; Electronic Journals of
Environmental, Agricultural and Food Chemistry; Newsline; Journal of Oil, Soap,
Cosmetics; Trends World Food; Trends Food Science & Technology; Pakistani Journal
of Nutrition; Journal of Food Technology; Academic Food; and Australian Journal of
Science & Technology. He is referee/reviewer for AOAC International, Journal of
Experimental Marine Biology and Ecology, Journal of Medical Foods, die Nahrung,
Journal of Alternative & Complementary Medicine, The Analyst, and Journal of
Agricultural and Food Chemistry.


Acknowledgments
Permission to reprint the following is gratefully acknowledged:
Table 4.1: Kolakowski, E., Protein determination and analysis in food systems, in
Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic
Publishing, Lancaster/Basel, chap. 4, pp. 57–112, 2001.
Figure 11.3: Orlandi, P.A. et al., Analysis of flour and food samples for Cry9C from
bioengineered corn, J. Food Prot., 65, 426, 2002.
Figure 11.4: Raybourne, R.B. et al., Development and use of an ELISA test to detect
IgE antibody to Cry9c following exposure to bioengineered corn, Int. Arch. Allergy
Immunol., 132(4), 322, 2003.

Contributors
Aldert A. Bergwerff
Utrecht University
Utrecht, The Netherlands
Marek Biziuk
Gdansk University of Technology
Gdansk, Poland
Richard Brereton
University of Bristol
Bristol, United Kingdom
Stephen G. Capar
U.S. Food and Drug Administration
College Park, Maryland
Francisco Diez-Gonzalez
University of Minnesota
St. Paul, Minnesota
Douglas G. Hayward
U.S. Food and Drug Administration
College Park, Maryland
Yildiz Karaibrahimoglu
U.S. Department of Agriculture
Wyndmoor, Pennsylvania

Jae Hwan Lee
Department of Food Science and
Technology
Seoul National University of
Technology
Seoul, Korea

Steven J. Lehotay
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Dan Levy
U.S. Food and Drug Administration
Laurel, Maryland
Kannapon Lopetcharat
Unilever Corporation
Edgewater, New Jersey
Katerina Mastovska
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Mina McDaniel
Oregon State University
Corvallis, Oregon

Edward Kolakowski
Agricultural University of Szczecin
Szczecin, Poland

Malgorzata Michalska
Institute of Maritime and Tropical
Medicine
Gdynia, Poland

Keith A. Lampel
U.S. Food and Drug Administration
Laurel, Maryland

Robert A. Moreau

U.S. Department of Agriculture
Wyndmoor, Pennsylvania


Marian Naczk
St. Francis Xavier University
Antigonish, Nova Scotia, Canada

Andras Szabo
Szent Istvan University
Budapest, Hungary

Palmer A. Orlandi
U.S. Food and Drug Administration
Laurel, Maryland

Piotr Szefer
Medical University of Gdansk
Gdansk, Poland

Semih Ötles¸
Ege University
Izmir, Turkey

Sandor Tarjan
National Food Control Institute
Budapest, Hungary

Richard B. Raybourne
U.S. Food and Drug Administration

Laurel, Maryland

Mary W. Trucksess
U.S. Food and Drug Administration
Laurel, Maryland

Adriaan Ruiter
Wageningen Agricultural University
Bilthoven, The Netherlands

Michael H. Tunick
U.S. Department of Agriculture
Wyndmoor, Pennsylvania

Steven J. Schwartz
Ohio State University
Columbus, Ohio

Carmen D. Westphal
U.S. Food and Drug Administration
Laurel, Maryland

Fereidoon Shahidi
Memorial University of Newfoundland
St. John’s, Newfoundland, Canada

Kristina M. Williams
U.S. Food and Drug Administration
Laurel, Maryland

Contents
Chapter 1
Selection of Techniques Used in Food Analysis ......................................................1
Michael H. Tunick
Chapter 2
Statistical Assessment of Results of Food Analysis ...............................................15
Richard Brereton
Chapter 3
Analysis of Drinking Water.....................................................................................31
⁄
Marek Biziuk and Malgorzata
Michalska
Chapter 4
Analysis of Proteins, Peptides, and Amino Acids in Foods ...................................59
Edward Kolakowski
Chapter 5
Extraction and Analysis of Food Lipids .................................................................97
Robert A. Moreau
Chapter 6
Determination and Speciation of Trace Elements in Foods .................................111
Stephen G. Capar and Piotr Szefer
Chapter 7
Analysis of Vitamins for the Health, Pharmaceutical, and Food Sciences ..........159
Semih Ötles¸ and Yildiz Karaibrahimoglu
Chapter 8
Analysis of Carotenoids and Chlorophylls in Foods............................................179
Jae Hwan Lee and Steven J. Schwartz

Chapter 9
Analysis of Polyphenols in Foods.........................................................................199
Fereidoon Shahidi and Marian Naczk
Chapter 10
Sensory Analysis of Foods ....................................................................................261
Kannapon Lopetcharat and Mina McDaniel
Chapter 11
Determination of Food Allergens and Genetically Modified
Components ...........................................................................................................303
Kristina M. Williams, Mary W. Trucksess, Richard B. Raybourne,
Palmer A. Orlandi, Dan Levy, Keith A. Lampel, and Carmen D. Westphal
Chapter 12
Determination of Pesticide Residues ....................................................................329
Steven J. Lehotay and Katerina Mastovska
Chapter 13
Determination of Pollutants in Foods ...................................................................361
Douglas G. Hayward
Chapter 14
Analysis of Chemical Preservatives in Foods.......................................................379
Adriaan Ruiter and Aldert A. Bergwerff
Chapter 15
Measuring Radioactive Contaminants in Foods ...................................................403
Andras Szabo and Sandor Tarjan
Chapter 16
Rapid Analysis Techniques in Food Microbiology...............................................415
Francisco Diez-Gonzalez and Yildiz Karaibrahimoglu
Index......................................................................................................................433


1

Selection of Techniques
Used in Food Analysis
Michael H. Tunick

CONTENTS
1.1
1.2
1.3
1.4
1.5

Introduction ......................................................................................................2
Sample Selection and Preservation..................................................................2
Extraction .........................................................................................................3
Technique Selection .........................................................................................4
Application of Techniques ...............................................................................4
1.5.1 Chromatographic Techniques................................................................5
1.5.1.1 Gas Chromatography (GC)......................................................5
1.5.1.2 High-Performance Liquid Chromatography (HPLC)..............5
1.5.1.3 Supercritical Fluid Chromatography (SFC) ............................6
1.5.2 Spectroscopic Techniques .....................................................................6
1.5.2.1 UV, Vis, and Fluorescence .......................................................6
1.5.2.2 Infrared (IR) .............................................................................6
1.5.2.3 Raman.......................................................................................7
1.5.2.4 Atomic Absorption and Atomic Emission...............................7
1.5.2.5 Mass Spectrometry (MS) .........................................................7
1.5.2.6 Nuclear Magnetic Resonance (NMR) and
Electron Spin Resonance (ESR) ..............................................8
1.5.2.7 Other Spectroscopic Techniques..............................................8

1.5.3 Physical Techniques ..............................................................................9
1.5.3.1 Electrochemical ........................................................................9
1.5.3.2 Electrophoresis .........................................................................9
1.5.3.3 Flavor and Odor .......................................................................9
1.5.3.4 Particle Analysis.....................................................................10
1.5.3.5 Rheology and Texture ............................................................10
1.5.3.6 Structure .................................................................................10
1.5.3.7 Thermal Properties.................................................................10
1.5.4 Biological Techniques .........................................................................11
1.5.4.1 Enzyme and Microbial Sensors .............................................11
1.5.4.2 Immunosensors.......................................................................11
1.6 Summary ........................................................................................................11
References................................................................................................................12

1


2

1.1

Methods of Analysis of Food Components and Additives

INTRODUCTION

Scientists analyze foods for their composition; structure; and chemical, physical,
and biological properties. The information obtained may be used for research or for
monitoring product quality. A host of different analyses can be conducted on any
food. For example, a cheese manufacturer or researcher could investigate the following:
Composition

• Proximate analysis (protein, phosphorus)
• Specific components (beta-casein, fat in dry matter)
Structure
• Macrostructure: Visible to naked eye (color, curd pieces)
• Microstructure: 0.1–100 ␮m range (protein matrix, fat globules)
• Ultrastructure: Nanometer range (casein micelles and submicelles)
Chemical and physical properties
• Flavor (bitter, salty)
• Odor (diacetyl, lactone)
• Rheology (hardness, elasticity)
• Stability (fat oxidation, whey leakage)
• Thermal properties (heat of combustion, melting profile)
Biological properties
• Growth of microorganisms (starter bacteria, mold)
• Metabolic processes and products (enzymes, peptides)
Sampling and method selection are of great importance in food analysis. Food
is heterogeneous, and changes due to age, physical handling, temperature, and other
factors will affect analytical results. Food is eaten for enjoyment as well as nutrition,
so techniques dealing with aroma, flavor, and texture should not be ignored. This
chapter will cover the most common methods used in food analysis, and will include
sample preparation and choice of technique.

1.2

SAMPLE SELECTION AND PRESERVATION

The first step in food analysis is sample selection. Ideally, a sample will be identical
to the material from which it has been removed. Samples can be chosen at random,
by judgment of the analyst, or according to a system based on timing or location
(such as daily at noon, or within a specific portion of the product or its container).

Samples must be representative, collected without contamination, and properly handled for the analytical results to be meaningful. If the analysis is not to be performed
immediately, the sample will probably have to be preserved to prevent deterioration.
Preservation involves the control of temperature, moisture, oxygen, and light, and


Selection of Techniques Used in Food Analysis

3

may be as simple as sealing the sample in a container and placing it in a refrigerator.
Containers must be dry, sterile, and unbreakable.

1.3

EXTRACTION

Food samples frequently have to undergo extraction, separation, or concentration
procedures prior to analysis. Foods contain a myriad of compounds and are not
homogeneous, often forcing the removal of interfering components and the isolation
of the analyte before an analysis is attempted. Common procedures include distillation, filtration, and precipitation. The sample may also have to be homogenized,
ground, or treated in some other way. Buldini et al.1 and Smith2 reviewed a number
of modern extraction techniques, which include the following:
Digestion
• Microwave oven digestion, with acids such as nitric or sulfuric, for
solubilizing and oxidizing organic compounds to obtain free ions.
Digestion by microwave is faster than the classical wet digestion.
• UV photolysis digestion, with hydrogen peroxide, for degrading
organic compounds with hydroxyl radicals to obtain free ions. Small
amounts of reagents are required, but digestion time is longer.
Membrane

• Microfiltration or ultrafiltration, based on size exclusion. Polymer
membranes are often used for separation.
• Dialysis, based on ionic charge and size exclusion. Cellulose membranes are typically used.
Solvent
• Solvent extraction, for dissolving compounds of interest.
• Pressurized fluid extraction, at the near-supercritical region, where
extraction is faster and more efficient.
• Supercritical fluid extraction, above the critical pressure and temperature of carbon dioxide, which is nontoxic and nonpolluting. Extraction
is completed in minutes instead of hours, and thermal degradation is
reduced.
• Microwave-assisted extraction, usually requiring Ͻ15 mL solvent,
Ͻ10 min extraction time, and no elevated pressure.
Sorbent
• Solid-phase extraction and microextraction, where analytes are held
by sorbents such as silica or polymers, and then solubilized and eluted.
Headspace
• Purge and trap (or dynamic headspace), in which the analyte is flushed
from a liquid or gaseous sample and concentrated in a cryogenic trap.


4

Methods of Analysis of Food Components and Additives

• Adsorbent trap, where a synthetic porous polymer adsorbs a gas which
is then desorbed.
The digestion methods are employed for extracting ions, and the other methods
are used to extract compounds. Dialysis is used for both. Some of the extraction
methods, such as supercritical fluid and sorbent, are used in conjunction with analytical techniques.

1.4

TECHNIQUE SELECTION

The analyst decides the problem to be solved and plans the analyses required,
choosing techniques for their appropriateness using criteria in a number of categories:
Ability to conduct analysis
• Sample size, reagents, instruments, cost, final state of sample
(destroyed or intact)
Fundamental characteristics
• Precision, accuracy, sensitivity, specificity, detection limit, reproducibility
Personnel concerns
• Safety, simplicity, speed
Technique status
• Official method, in-house method
Official methods are developed by being comprehensively studied and compared
between laboratories. Standardized official methods include those published by
AOAC International,3 American Association of Cereal Chemists,4 and American Oil
Chemists’ Society.5 More specialized method collections, such as Food Chemical
Codex6 for determination of additives, have also been compiled. Many techniques
are not listed as official because they are relatively new or have not yet been applied
to certain types of samples. In these cases, in-house methods may be used if they
have been validated. A review of validation methods was published by Wood.7

1.5

APPLICATION OF TECHNIQUES

A multitude of analytical techniques are available for food. Many gravimetric and
titrimetric methods are well established and will not be discussed here. The number

of instrumental methods has been steadily growing, and can be broadly categorized
as chromatographic, spectroscopic, physical, and biological.


Selection of Techniques Used in Food Analysis

1.5.1

5

CHROMATOGRAPHIC TECHNIQUES

Chromatography is based on distribution or partition of a sample solute between
stationary and mobile phases. Chromatographic techniques in common use today in
food analysis include gas chromatography (GC), high-performance liquid chromatography (HPLC), and supercritical fluid chromatography (SFC). These often serve
as a separation method when connected to another instrument such as a mass
spectrometer, which serves as the detector.
1.5.1.1

Gas Chromatography (GC)

GC was introduced in the 1950s and has been applied to a wide range of foods. It
is applicable to volatile substances that are thermally stabile; LC and SFC are more
appropriate chromatographic methods for analysis of amino acids, peptides, sugars,
and vitamins. GC is useful for analysis of nonpolar compounds, although polar
compounds may be analyzed if derivatized first. Isolation of the analyte from the
sample matrix is particularly important in GC to avoid false responses from matrix
degradation products. Headspace methods (including direct sampling of the headspace), distillation, and solvent extraction are often employed. Detectors include
thermal conductivity (which is nonspecific), flame ionization (for most organic
compounds), electron capture (mainly for pesticide residues), and flame photometric

(for pesticides and sulfur compounds). The most common food analysis applications
for GC involve carbohydrates, drugs, lipids, and pesticides.8
Improvements in chromatography are constantly occurring. For instance, a new
approach is comprehensive chromatography, which allows a sample to be separated
along two independent axes. Comprehensive two-dimensional gas chromatography,
GC ϫ GC, consists of a high-resolution column with a nonpolar stationary phase,
a modulator for separating the eluate into many small fractions, and a second column
which is short, narrow, and polar. This technique has been applied to fatty acids,
flavors, and pesticides, and was reviewed by Dallüge et al.9
1.5.1.2

High-Performance Liquid Chromatography (HPLC)

HPLC was developed in the 1960s as an improvement over column liquid chromatography and has been used to measure nonvolatile food components. Spectroscopic
detectors are often employed. Normal-phase HPLC, in which the stationary phase
is a polar adsorbent and the mobile phase is a nonpolar solvent, is often used for
fat-soluble vitamins and carbohydrates. Reversed-phase HPLC, with a nonpolar
stationary phase and polar mobile phase, is more popular because of its wider
application. Ion-exchange HPLC, with a functionalized organic resin as packing
material, is used for detection of inorganic ions and analysis of carbohydrates and
amino acids. HPLC is currently the most popular food analysis technique (GC is
second) and is most used for amino acids, carbohydrates, drugs, lipids, and proteins.8
A new application of this technique is comprehensive two-dimensional liquid
chromatography ϫ gas chromatography, LC ϫ GC. Triglycerides can first be


6

Methods of Analysis of Food Components and Additives

separated according to double bond content and then by carbon number. Janssen et
al.10 demonstrated fingerprinting of olive oil, which can be applied to place of origin
analysis, by separation into mono-, di-, and triglycerides as well as sterols, esters,
and other compound classes.
1.5.1.3

Supercritical Fluid Chromatography (SFC)

Supercritical carbon dioxide serves as the mobile phase in SFC; an open tubular
column or a packed column is employed as the stationary phase, and any GC or LC
detector is used. Instrumentation first became available in the 1980s. Smith11
reviewed the history and applications of supercritical fluids, citing its use in separating lipids from food matrices as a chief advantage over other methods. However,
SFC is prone to operational difficulties and is a normal-phase method; reversedphase HPLC is often viewed as preferable.

1.5.2

SPECTROSCOPIC TECHNIQUES

Spectroscopy is based on interactions of matter with electromagnetic radiation.
Interactions can take the form of absorption and emission, and can be detected by
using emission, transmission, and reflection designs. Food scientists most often deal
with the ultraviolet (UV), visible (Vis), infrared (IR), radio (nuclear magnetic resonance, NMR), and microwave (electron spin resonance, ESR) regions of the spectrum, and use spectroscopic techniques for quantitative and qualitative analyses.
1.5.2.1

UV, Vis, and Fluorescence

UV and Vis spectroscopy measure absorbed radiation and have been used in food
laboratories for many years. A food component that absorbs in the ultraviolet or
visible range may be analyzed at its characteristic wavelength in a UV-Vis spectrophotometer, as long as there are no interfering compounds.
Fluorescence spectroscopy deals with emitted radiation, and can be three orders

of magnitude more sensitive than UV or Vis spectroscopy. Many organic molecules
fluoresce, including bacteria and some pesticide residues, making fluorescence spectroscopy an option for detecting food contamination.
1.5.2.2

Infrared (IR)

Many molecular groups absorb IR light at specific wavelengths in an infrared
spectrum, with the fingerprint region of the spectrum leading to positive identification
of compounds. This long-standing technique has been expanded upon in recent years.
Fourier transform infrared (FTIR) spectrometers are now used on production lines
for determining concentrations of fat, protein, and moisture. This on-line method of
analysis has a large advantage over other techniques because the sample does not
have to be extracted or treated in any way. Attenuated total reflectance (ATR) deals
with internal reflection of IR light, and it has been used to examine sugars and trans
fatty acids. High-pressure and high-temperature ATR cells have been developed.
This technique can be enhanced by using multiple internal reflection (MIR), in which


Selection of Techniques Used in Food Analysis

7

light is bounced off the surface several times. Further developments in the optical
components are needed before this method can be used more extensively on foods.
The newest IR technique is diffuse reflectance infrared Fourier transform (DRIFT),
which measures the sum of surface-reflected light and light that has been absorbed
and reemitted. DRIFT has been employed recently to monitor production and detect
compounds in coffee. Recent developments in IR spectroscopy of foods have been
reviewed by Wilson and Tapp.12
1.5.2.3

Raman

Raman spectroscopy is a complementary technique to IR spectroscopy. IR absorption
depends on changes in dipole moment, meaning that polar groups have strong IR
responses. Raman scattering deals with changes in polarizability of functional
groups, so nonpolar groups produce intense responses. Proteins and amino acids
lend themselves to Raman spectroscopy, and carbohydrates, lipids, and minor food
components are also examined by this technique. In addition to basic research on
molecular structure, Raman spectroscopy is now being used for industrial process
control. Li-Chan reviewed the application of Raman spectroscopy for food analysis.13
1.5.2.4

Atomic Absorption and Atomic Emission

Atomic absorption spectroscopy (AAS) is based on absorption of UV-Vis radiation
by atomized minerals, whereas atomic emission spectroscopy (AES) uses the emission of radiation by a sample. Samples must usually be ashed, dissolved in water
or dilute acid, and vaporized. In AAS, samples are atomized by nebulizer and burner
(flame AAS), or by a graphite furnace (electrothermal AAS). Electrothermal AAS
uses smaller samples and has much lower detection limits than flame AAS, but it is
more costly and less precise. In AES, atomization and excitation can be performed
by flame or by inductively coupled plasma (ICP), where samples are heated to over
6000 K in the presence of argon. Both AAS and AES measure trace metal concentrations in complex matrices with excellent precision and accuracy. AAS is the more
established technique, with a wider variety of instruments available, but ICP-AES
can be used to measure more than one element in a sample and can measure
compounds that are stable at high temperatures. Both AAS and AES have supplanted
classical methods for detecting minerals in food.
1.5.2.5

Mass Spectrometry (MS)

A mass spectrometer ionizes molecules to produce charged fragments that are
separated by size and charge. MS has been used for identification and analysis of
complex compounds since the early 1960s. The coupling of separation techniques
with MS, which began in the 1970s, has overcome the main analytical problem with
chromatographic techniques — namely, ambiguity about the identity of the analyte.
MS is frequently used in combination with GC, HPLC, ICP, and capillary electrophoresis, and there are tandem MS-MS instruments. Three new ionization techniques
used in food analysis are electrospray ionization (ESI, where multiply charged ions
are produced by repeated formation and explosion of charged droplets), heated


8

Methods of Analysis of Food Components and Additives

nebulizer-atmospheric pressure chemical ionization (HN-APCI, where a gas-phase
ion-molecule reaction process allows the analyte molecules to be ionized under
atmospheric pressure), and matrix-assisted laser desorption/ionization (MALDI,
where a sample is crystallized in a matrix of small aromatic compounds, and the
crystal is subjected to a pulsed ultraviolet laser that fragments the molecules). MS
techniques have been used to analyze the gamut of food components, including
antioxidants, aroma compounds, carbohydrates, drug residues, lipids, peptides and
proteins, toxins, and vitamins. A summary of developments in the use of MS in food
analysis was published by Careri et al.14
1.5.2.6

Nuclear Magnetic Resonance (NMR) and Electron Spin
Resonance (ESR)

NMR is a spectroscopic method in which atomic nuclei that are oriented by a

magnetic field absorb characteristic frequencies in the radio range. ESR deals with
electrons and microwave frequencies. These techniques have several advantages:
they are nondestructive, do not usually require sample separation or extraction, and
can analyze the interior of a sample. Drawbacks include lower sensitivity and
selectivity than some other techniques. NMR experiments are performed using
continuous wave (magnetic field held constant and oscillating frequency varied, or
vice versa) or pulse (short time, large amplitude) methods; ESR uses continuous
wave. Available NMR instruments include low-resolution (for moisture or oil content), high-resolution liquid (analysis of liquid phase), high-resolution solid (analysis
of solid phase), and magnetic resonance imaging (three-dimensional views of cross
sections of foods).
Virtually any food can be analyzed by NMR and ESR. NMR is often used to
examine physical properties such as melting, crystallization, polymorphism, and oil
content, and ESR is used for detecting free radicals produced in physical and
chemical processes. Mannina et al. summarized the principles of NMR and applied
the technique to analyzing free acidity, fatty acid profile, and sterol, squalene, and
chlorophyll content as methods of authenticating olive oil.15
1.5.2.7

Other Spectroscopic Techniques

Consumers rely on color, flavor, odor, and texture to determine the quality of food.
Colorimeters are used to qualitate and quantitate food color, with measurements
based on hue, lightness, and saturation, and are often used in conjunction with
sensory and shelf-life studies. A digital camera and computer graphics software have
recently been applied to the analysis of surface color of food.16
Refractometry is based on the change in velocity of light by the analyte. Refractive index measurements are useful in determining concentrations of beverages,
sauces, and other liquid foods. HPLC instruments sometimes have refractometer
detectors.
Polarimetry is the study of the rotation of polarized light by optically active
substances. Polarimetry is used to distinguish optical isomers, identify and characterize optically active substances, and measure their change in concentration during

Selection of Techniques Used in Food Analysis

9

reactions. It is commonly applied to the measurement of oils in the flavor industry,
sugars, and starches. Circular dichroism and optical rotatory dispersion are based
on the interaction of circularly polarized light with optically active species; the
former depends on wavelength and the latter on molar absorptivity. These techniques
are often applied to amino acids, peptides, proteins, and complex natural products.
Ultrasonic sensors have been applied to the determination of compositional and
textural properties by measurement of velocity of ultrasound waves through a sample. Ultrasonic imaging is used to examine structure in foods, but is too timeconsuming for routine inspections. Coupland and Saggin17 summarized the use of
ultrasonic sensors in food analysis.

1.5.3
1.5.3.1

PHYSICAL TECHNIQUES
Electrochemical

The most common electrochemical technique is the familiar pH electrode. An alternative to AAS and AES is the ion-selective electrode, which is sensitive to a particular
ion. This technique is simple, rapid, and relatively inexpensive. However, these
electrodes are not ion-specific, as there may be interference from ions other than
those being examined.
1.5.3.2

Electrophoresis

The basis for gel electrophoresis, developed in the 1950s, is the separation of charged

molecules when an electric field is applied. The main types of electrophoresis are
nondenaturing, where separation is according to charge, shape, and size; denaturing
or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), where
the separation is primarily by molecular weight; isoelectric focusing, which separates
by charge; and two-dimensional, with separations in perpendicular directions by
isoelectric focusing and SDS-PAGE. The analysis is carried out on a porous gel and
is mainly used for studying nucleic acids and proteins.
Capillary electrophoresis (CE), developed in the 1980s, uses a capillary tube
and photometric detection. CE techniques include capillary zone electrophoresis,
for charged analytes, and micellar electrokinetic chromatography, for neutral analytes. CE has been applied to analysis of amino acids, carbohydrates, proteins, and
vitamins, as well as additives, natural toxins, and antibiotic and pesticide residues.
Detection limits are relatively high, however, because of the low sample volume.
Dong18 reviewed CE in the analysis of food.
1.5.3.3

Flavor and Odor

No food will be eaten if its aroma and flavor are unacceptable, so characterizing
these attributes is important to the food industry. Sensory panels are often used, but
since they rely on human judgment, instrumental techniques are being developed.
Some 7000 aroma compounds have been identified by GC-MS. The sensory character of individual aroma compounds is often investigated by gas chromatographyolfactometry (GC-O), developed in the 1970s, where analysts sniff compounds as


10

Methods of Analysis of Food Components and Additives

they elute from a GC. Atmospheric pressure ionization-mass spectroscopy (APIMS) measures the concentration of volatiles as they are being inhaled, providing
information on flavor release. New advances in these areas were described by Risch
and Ho,19 and Marsili20 covered sample preparation, instrumental techniques, and

applications. One challenge to be overcome in characterizing flavors and odors is
that individual components are analyzed separate from the food matrix, and therefore
out of context.
1.5.3.4

Particle Analysis

Many processed foods contain particles produced by drying, grinding, milling, or
other means. Particle appearance and shape are examined by the optical microscope,
and size uniformity is measured by particle sizing instruments based on principles
such as laser diffraction and light scattering.21
1.5.3.5

Rheology and Texture

Rheology is the study of the flow and deformation of matter, and texture deals with
a consumer’s perception of rheology. Rheology is a factor in food process engineering, shelf-life testing, and quality control. Food exhibits both elastic and viscous
behavior, which can be measured by viscometric, oscillatory shear, compression,
extension, torsion, and other tests. Bourne22 described rheological and textural measurement of food.
1.5.3.6

Structure

Food structure is studied by light microscopy, scanning electron microscopy (SEM),
transmission electron microscopy (TEM), and confocal laser scanning microscopy
(CLSM). Light microscopy, with up to ϫ2000 magnification, is used to survey
structural details. Surface microstructure is examined through SEM, and internal
structures are visualized by TEM. CLSM, which requires less sample preparation,
is used to obtain three-dimensional images. Kalab23 maintains a Web site devoted
to food microscopy.

1.5.3.7

Thermal Properties

Thermal transitions in food such as melting, decomposition, and glass transitions
are observed by using a differential scanning calorimeter, in which a sample is heated
and the amount of heat absorbed relative to a reference is measured. The technique
has been applied to components such as proteins, starches, and sugars, and is
especially useful for observing the melting of lipids, which have relatively high heats
of fusion. Applications of thermal analysis in food have been reviewed by Harwalkar
and Ma.24
Bomb calorimeters are used to determine the caloric value of a food by combusting it in an oxygen atmosphere and measuring the temperature change in the


Selection of Techniques Used in Food Analysis

11

surrounding water. However, many manufacturers obtain caloric values by simple
calculation, using percentages of each ingredient and caloric values for fat, carbohydrate, and protein.

1.5.4

BIOLOGICAL TECHNIQUES

1.5.4.1

Enzyme and Microbial Sensors

Biosensors consist of a biological recognition element that produces a quantifiable

response in a signal transduction element when in contact with the analyte. Enzyme
biosensors use enzymes to generate products that are detected by acoustic, electrochemical, optical, and photothermal transduction elements. Microbial biosensors use
genetically modified microorganisms that are immobilized on a membrane or trapped
in a matrix, with the transduction mechanism consisting of an oxygen or pH electrode, or a luminometer if luciferin is added. A popular type of optical element
developed in the 1990s is surface plasmon resonance.
These types of biosensors are often applied to detection of contaminants such
as herbicides, pesticides, pathogens, and toxins, as well as food components such
as carbohydrates and amino acids. Improvements in response time, sensitivity, and
specificity are needed for wider acceptance of this technique. Recent reviews by
Patel25 and Mello and Kabota26 describe biosensors and their suitability in food
analysis.
1.5.4.2

Immunosensors

Immunosensors are biosensors in which the biological recognition elements are
antibodies that are attached to a solid support and bind to a particular antigen or
antibody in the sample. The most common immunoassay is enzyme-linked immunosorbent assay (ELISA), in which an enzyme-linked antibody is applied after the
antigen or antibody is bound. A substrate is then added to produce a secondary
reaction that has a colored product that is measured spectroscopically. The antibody/antigen interaction is specific enough to allow detection of species of origin,
and it is also used to detect allergens, enzymatic inactivation, genetically modified
organisms, microbial contamination, and toxins.

1.6

SUMMARY

The analytical techniques now available to food researchers provide faster results at
lower cost with lower solvent and reagent use and higher precision and accuracy
than classical methods. Choosing the appropriate method requires the scientist to

be aware of its strengths and limitations. When the technique is successfully applied,
a wealth of information on composition, properties, and structure of food and food
components can be uncovered.


12

Methods of Analysis of Food Components and Additives

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