Flavour, fragrance and odor analysis

Marcel Dekker, Inc. New York
•
Basel
TM
FLAVOR,
FRAGRANCE,
and ODOR
ANALYSIS
edited by
Ray Marsili
Dean Foods Company
Rockford, Illinois
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
ISBN: 0-8247-0627-7
This book is printed on acid-free paper.
Headquarters
Marcel Dekker, Inc.
270 Madison Avenue, New York, NY 10016
tel: 212-696-9000; fax: 212-685-4540
Eastern Hemisphere Distribution
Marcel Dekker AG
Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland
tel: 41-61-261-8482; fax: 41-61-261-8896
World Wide Web
http:/ /www.dekker.com
The publisher offers discounts on this book when ordered in bulk quantities. For more
information, write to Special Sales/Professional Marketing at the headquarters address
above.
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
Neither this book nor any part may be reproduced or transmitted in any form or by any

means, electronic or mechanical, including photocopying, microfilming, and recording,
or by any information storage and retrieval system, without permission in writing from
the publisher.
Current printing (last digit):
10987654321
PRINTED IN THE UNITED STATES OF AMERICA
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
FOOD SCIENCE AND TECHNOLOGY
A Series of Monographs, Textbooks, and Reference Books
EDITORIAL BOARD
Senior Editors
Owen R. Fennema University of Wisconsin–Madison
Y.H. Hui Science Technology System
Marcus Karel Rutgers University (emeritus)
Pieter Walstra Wageningen University
John R. Whitaker University of California–Davis
Additives P. Michael Davidson University of Tennessee–Knoxville
Dairy science James L. Steele University of Wisconsin–Madison
Flavor chemistry and sensory analysis John H. Thorngate III University
of California–Davis
Food engineering Daryl B. Lund University of Wisconsin–Madison
Food proteins/food chemistry Rickey Y. Yada University of Guelph
Health and disease Seppo Salminen University of Turku, Finland
Nutrition and nutraceuticals Mark Dreher Mead Johnson Nutritionals
Phase transition/food microstructure Richard W. Hartel University of
Wisconsin–Madison
Processing and preservation Gustavo V. Barbosa-Cánovas Washington
State University–Pullman
Safety and toxicology Sanford Miller University of Texas–Austin
1. Flavor Research: Principles and Techniques, R. Teranishi, I. Horn-

stein, P. Issenberg, and E. L. Wick
2. Principles of Enzymology for the Food Sciences, John R. Whitaker
3. Low-Temperature Preservation of Foods and Living Matter, Owen R.
Fennema, William D. Powrie, and Elmer H. Marth
4. Principles of Food Science
Part I: Food Chemistry, edited by Owen R. Fennema
Part II: Physical Methods of Food Preservation, Marcus Karel, Owen
R. Fennema, and Daryl B. Lund
5. Food Emulsions, edited by Stig E. Friberg
6. Nutritional and Safety Aspects of Food Processing, edited by Steven
R. Tannenbaum
7. Flavor Research: Recent Advances, edited by R. Teranishi, Robert A.
Flath, and Hiroshi Sugisawa
8. Computer-Aided Techniques in Food Technology, edited by Israel
Saguy
9. Handbook of Tropical Foods, edited by Harvey T. Chan
10. Antimicrobials in Foods, edited by Alfred Larry Branen and P. Michael
Davidson
11. Food Constituents and Food Residues: Their Chromatographic
Determination, edited by James F. Lawrence
12. Aspartame: Physiology and Biochemistry, edited by Lewis D. Stegink
and L. J. Filer, Jr.
13. Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects,
edited by Lawrence J. Machlin
14. Starch Conversion Technology, edited by G. M. A. van Beynum and J.
A. Roels
15. Food Chemistry: Second Edition, Revised and Expanded, edited by
Owen R. Fennema
16. Sensory Evaluation of Food: Statistical Methods and Procedures, Mi-
chael O'Mahony

17. Alternative Sweeteners, edited by Lyn O'Brien Nabors and Robert C.
Gelardi
18. Citrus Fruits and Their Products: Analysis and Technology, S. V. Ting
and Russell L. Rouseff
19. Engineering Properties of Foods, edited by M. A. Rao and S. S. H.
Rizvi
20. Umami: A Basic Taste, edited by Yojiro Kawamura and Morley R.
Kare
21. Food Biotechnology, edited by Dietrich Knorr
22. Food Texture: Instrumental and Sensory Measurement, edited by
Howard R. Moskowitz
23. Seafoods and Fish Oils in Human Health and Disease, John E.
Kinsella
24. Postharvest Physiology of Vegetables, edited by J. Weichmann
25. Handbook of Dietary Fiber: An Applied Approach, Mark L. Dreher
26. Food Toxicology, Parts A and B, Jose M. Concon
27. Modern Carbohydrate Chemistry, Roger W. Binkley
28. Trace Minerals in Foods, edited by Kenneth T. Smith
29. Protein Quality and the Effects of Processing, edited by R. Dixon
Phillips and John W. Finley
30. Adulteration of Fruit Juice Beverages, edited by Steven Nagy, John A.
Attaway, and Martha E. Rhodes
31. Foodborne Bacterial Pathogens, edited by Michael P. Doyle
32. Legumes: Chemistry, Technology, and Human Nutrition, edited by
Ruth H. Matthews
33. Industrialization of Indigenous Fermented Foods, edited by Keith H.
Steinkraus
34. International Food Regulation Handbook: Policy · Science · Law,
edited by Roger D. Middlekauff and Philippe Shubik
35. Food Additives, edited by A. Larry Branen, P. Michael Davidson, and

Seppo Salminen
36. Safety of Irradiated Foods, J. F. Diehl
37. Omega-3 Fatty Acids in Health and Disease, edited by Robert S. Lees
and Marcus Karel
38. Food Emulsions: Second Edition, Revised and Expanded, edited by
Kåre Larsson and Stig E. Friberg
39. Seafood: Effects of Technology on Nutrition, George M. Pigott and
Barbee W. Tucker
40. Handbook of Vitamins: Second Edition, Revised and Expanded,
edited by Lawrence J. Machlin
41. Handbook of Cereal Science and Technology, Klaus J. Lorenz and
Karel Kulp
42. Food Processing Operations and Scale-Up, Kenneth J. Valentas,
Leon Levine, and J. Peter Clark
43. Fish Quality Control by Computer Vision, edited by L. F. Pau and R.
Olafsson
44. Volatile Compounds in Foods and Beverages, edited by Henk Maarse
45. Instrumental Methods for Quality Assurance in Foods, edited by
Daniel Y. C. Fung and Richard F. Matthews
46. Listeria, Listeriosis, and Food Safety, Elliot T. Ryser and Elmer H.
Marth
47. Acesulfame-K, edited by D. G. Mayer and F. H. Kemper
48. Alternative Sweeteners: Second Edition, Revised and Expanded, ed-
ited by Lyn O'Brien Nabors and Robert C. Gelardi
49. Food Extrusion Science and Technology, edited by Jozef L. Kokini,
Chi-Tang Ho, and Mukund V. Karwe
50. Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee
51. Handbook of Food Engineering, edited by Dennis R. Heldman and
Daryl B. Lund
52. Food Analysis by HPLC, edited by Leo M. L. Nollet

53. Fatty Acids in Foods and Their Health Implications, edited by Ching
Kuang Chow
54. Clostridium botulinum: Ecology and Control in Foods, edited by
Andreas H. W. Hauschild and Karen L. Dodds
55. Cereals in Breadmaking: A Molecular Colloidal Approach,
Ann-Charlotte Eliasson and Kåre Larsson
56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul
57. Antimicrobials in Foods: Second Edition, Revised and Expanded,
edited by P. Michael Davidson and Alfred Larry Branen
58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright
59. Rice Science and Technology, edited by Wayne E. Marshall and
James I. Wadsworth
60. Food Biosensor Analysis, edited by Gabriele Wagner and George G.
Guilbault
61. Principles of Enzymology for the Food Sciences: Second Edition, John
R. Whitaker
62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C.
Akoh and Barry G. Swanson
63. Engineering Properties of Foods: Second Edition, Revised and
Expanded, edited by M. A. Rao and S. S. H. Rizvi
64. Handbook of Brewing, edited by William A. Hardwick
65. Analyzing Food for Nutrition Labeling and Hazardous Contaminants,
edited by Ike J. Jeon and William G. Ikins
66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar
G. Gaonkar
67. Food Polysaccharides and Their Applications, edited by Alistair M.
Stephen
68. Safety of Irradiated Foods: Second Edition, Revised and Expanded, J.
F. Diehl
69. Nutrition Labeling Handbook, edited by Ralph Shapiro

70. Handbook of Fruit Science and Technology: Production, Composition,
Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam
71. Food Antioxidants: Technological, Toxicological, and Health Perspec-
tives, edited by D. L. Madhavi, S. S. Deshpande, and D. K. Salunkhe
72. Freezing Effects on Food Quality, edited by Lester E. Jeremiah
73. Handbook of Indigenous Fermented Foods: Second Edition, Revised
and Expanded, edited by Keith H. Steinkraus
74. Carbohydrates in Food, edited by Ann-Charlotte Eliasson
75. Baked Goods Freshness: Technology, Evaluation, and Inhibition of
Staling, edited by Ronald E. Hebeda and Henry F. Zobel
76. Food Chemistry: Third Edition, edited by Owen R. Fennema
77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L.
Nollet
78. Computerized Control Systems in the Food Industry, edited by Gauri
S. Mittal
79. Techniques for Analyzing Food Aroma, edited by Ray Marsili
80. Food Proteins and Their Applications, edited by Srinivasan Damo-
daran and Alain Paraf
81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig
E. Friberg and Kåre Larsson
82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cánovas,
Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson
83. Milk and Dairy Product Technology, Edgar Spreer
84. Applied Dairy Microbiology, edited by Elmer H. Marth and James L.
Steele
85. Lactic Acid Bacteria: Microbiology and Functional Aspects: Second
Edition, Revised and Expanded, edited by Seppo Salminen and Atte
von Wright
86. Handbook of Vegetable Science and Technology: Production,
Composition, Storage, and Processing, edited by D. K. Salunkhe and

S. S. Kadam
87. Polysaccharide Association Structures in Food, edited by Reginald H.
Walter
88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by
Casimir C. Akoh and David B. Min
89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa
90. Dairy Technology: Principles of Milk Properties and Processes, P.
Walstra, T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van
Boekel
91. Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstätter
92. Listeria, Listeriosis, and Food Safety: Second Edition, Revised and
Expanded, edited by Elliot T. Ryser and Elmer H. Marth
93. Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho,
Leon Prosky, and Mark Dreher
94. Handbook of Food Preservation, edited by M. Shafiur Rahman
95. International Food Safety Handbook: Science, International Regula-
tion, and Control, edited by Kees van der Heijden, Maged Younes,
Lawrence Fishbein, and Sanford Miller
96. Fatty Acids in Foods and Their Health Implications: Second Edition,
Revised and Expanded, edited by Ching Kuang Chow
97. Seafood Enzymes: Utilization and Influence on Postharvest Seafood
Quality, edited by Norman F. Haard and Benjamin K. Simpson
98. Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D.
Todd
99. Handbook of Cereal Science and Technology: Second Edition, Re-
vised and Expanded, edited by Karel Kulp and Joseph G. Ponte, Jr.
100. Food Analysis by HPLC: Second Edition, Revised and Expanded,
edited by Leo M. L. Nollet
101. Surimi and Surimi Seafood, edited by Jae W. Park
102. Drug Residues in Foods: Pharmacology, Food Safety, and Analysis,

Nickos A. Botsoglou and Dimitrios J. Fletouris
103. Seafood and Freshwater Toxins: Pharmacology, Physiology, and
Detection, edited by Luis M. Botana
104. Handbook of Nutrition and Diet, Babasaheb B. Desai
105. Nondestructive Food Evaluation: Techniques to Analyze Properties
and Quality, edited by Sundaram Gunasekaran
106. Green Tea: Health Benefits and Applications, Yukihiko Hara
107. Food Processing Operations Modeling: Design and Analysis, edited
by Joseph Irudayaraj
108. Wine Microbiology: Science and Technology, Claudio Delfini and
Joseph V. Formica
109. Handbook of Microwave Technology for Food Applications, edited by
Ashim K. Datta and Ramaswamy C. Anantheswaran
110. Applied Dairy Microbiology: Second Edition, Revised and Expanded,
edited by Elmer H. Marth and James L. Steele
111. Transport Properties of Foods, George D. Saravacos and Zacharias
B. Maroulis
112. Alternative Sweeteners: Third Edition, Revised and Expanded, edited
by Lyn O’Brien Nabors
113. Handbook of Dietary Fiber, edited by Susan Sungsoo Cho and Mark
L. Dreher
114. Control of Foodborne Microorganisms, edited by Vijay K. Juneja and
John N. Sofos
115. Flavor, Fragrance, and Odor Analysis, edited by Ray Marsili
116. Food Additives: Second Edition, Revised and Expanded, edited by A.
Larry Branen, P. Michael Davidson, Seppo Salminen, and John H.
Thorngate, III
117. Food Lipids: Chemistry, Nutrition, and Biotechnology: Second Edition,
Revised and Expanded, edited by Casimir C. Akoh and David B. Min
118. Food Protein Analysis: Quantitative Effects on Processing, R. K.

Owusu-Apenten
119. Handbook of Food Toxicology, S. S. Deshpande
120. Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma, J.
Richard Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca
121. Physical Chemistry of Foods, Pieter Walstra
122. Handbook of Food Enzymology, edited by John R. Whitaker, Alphons
G. J. Voragen, and Dominic W. S. Wong
123. Postharvest Physiology and Pathology of Vegetables: Second Edition,
Revised and Expanded, edited by Jerry A. Bartz and Jeffrey K. Brecht
124. Characterization of Cereals and Flours: Properties, Analysis, and Ap-
plications, edited by Gönül Kaletunç and Kenneth J. Breslauer
125. International Handbook of Foodborne Pathogens, edited by Marianne
D. Miliotis and Jeffrey W. Bier
Additional Volumes in Preparation
Handbook of Dough Fermentations, edited by Karel Kulp and Klaus
Lorenz
Extraction Optimization in Food Engineering, edited by Constantina
Tzia and George Liadakis
Physical Principles of Food Preservation: Second Edition, Revised
and Expanded, Marcus Karel and Daryl B. Lund
Handbook of Vegetable Preservation and Processing, edited by Y. H.
Hui, Sue Ghazala, Dee M. Graham, K. D. Murrell, and Wai-Kit Nip
Food Process Design, Zacharias B. Maroulis and George D.
Saravacos
To Deb
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
Equipped with his five senses, man explores the universe around him
and calls the adventure science.
Edwin P. Hubble
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.

Preface
This book focuses on recent sample preparation techniques for isolating and con-
centrating flavor and odor chemicals from various types of foods, beverages, and
consumer products prior to gas chromatography (GC)/mass spectrometry (MS)
analysis. No single sample preparation technique is appropriate for every type
of analyte or matrix. We show the advantages, disadvantages, and biases of the
most common analytical techniques for flavor, fragrance, and odor analysis. The
intent of this book is to help chemists working with flavor, fragrance, and odor
problems to select the most appropriate techniques for studying specific applica-
tions. This text explores the application potential of various analytical techniques,
including numerous practical examples and tips that explain how state-of-the-art
techniques can be used to resolve important flavor, fragrance, and odor issues
facing chemists in the food and beverage and consumer product industries.
The book can be categorized into three parts: sample preparation and instru-
mentation techniques, application examples, and olfactometry. The final two
chapters discuss MS-based electronic nose applications (Chapter 13) and the
chemical structures of flavor and off-flavor chemicals in various types of foods
(Chapter 14).
This book follows up Techniques for Analyzing Food Aroma, and a few
chapters that discuss standard GC/MS sample preparation techniques have been
taken from that work. Some of the analytical techniques discussed in Techniques
for Analyzing Food Aroma were well established, while techniques such as solid-
phase microextraction (SPME) and electronic-nose applications were emerging
technologies. In recent years, more and more researchers have discoverd the nu-
merous advantages of SPME, and its popularity and use in extracting and concen-
trating flavor/odor-contributing analytes have skyrocketed. Several chapters in
this book emphasize SPME techniques.
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
For various reasons, reliable electronic-nose applications have been slower
to develop. Chapter 13 discusses the benefits of MS as a potential e-nose sensor.

This book also discusses the value of time-of-flight MS to the study of flavors
and odors. Incorporating the human sense of smell with potent analytical systems
is invaluable in problem solving. Just as sample preparation procedures and ana-
lytical instrumentation have continued to evolve and improve, so have olfactome-
try techniques. Chapters 11 and 12 cover various olfactometry techniques, includ-
ing a new, easier-to-implement method called SNIF.
I commend the contributing authors for their dedication, persistence, and
cooperation in completing their chapters in a timely manner. Unquestionably, the
information they have provided in this book, as well as in past publications, will
contribute greatly to the advancement of flavor and fragrance research. I would
also like to acknowledge my wife, Deborah, for her review of the chapters, her
patience, and her continuous support and words of encouragement.
Ray Marsili
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
Contents
Preface
Contributors
1.SolventExtractionandDistillationTechniques
Thomas Parliment
2. Analysis of Food Volatiles Using Headspace-Gas
ChromatographicTechniques
Thomas P. Wampler
3. The Analysis of Food Volatiles Using Direct Thermal
Desorption
Casey C. Grimm, Steven W. Lloyd, James A. Miller, and Arthur
M. Spanier
4.Solid-PhaseMicroextractionfortheAnalysisofAromasand
Flavors
Alan D. Harmon
5.TheAdvantagesofGC-TOFMSforFlavorandFragrance

Analysis
John F. Holland and Ben D. Gardner
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
6. Modern Methods for Isolating and Quantifying Volatile Flavor
andFragranceCompounds
Peter Werkhoff, Stefan Brennecke, Wilfried Bretschneider, and
Heinz-Ju
¨
rgen Bertram
7.SPMEComparisonStudiesandWhatTheyReveal
Ray Marsili
8. Analysis of Volatile Compounds in the Headspace of Rice
UsingSPME/GC/MS
Casey C. Grimm, Elaine T. Champagne, and Ken’ichi Ohtsubo
9. Headspace Techniques for the Reconstitution of Flower Scents
andIdentificationofNewAromaChemicals
Thomas McGee and Kenneth L. Purzycki
10.SPMEApplicationsinConsumerProducts
Richard Payne, Allen E. Puchalski, and John Labows
11.GasChromatography–OlfactometryinFoodAromaAnalysis
Imre Blank
12. Quantitative Use of Gas Chromatography–Olfactometry: The
GC-‘‘SNIF’’Method
Alain Chaintreau
13. Combining Mass Spectrometry and Multivariate Analysis to
MakeaReliableandVersatileElectronicNose
Ray Marsili
14.CharacterImpactCompounds:FlavorsandOff-FlavorsinFoods
Robert J. McGorrin
Abbreviations

Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
Contributors
Heinz-Ju
¨
rgen Bertram Haarmann & Reimer GmbH, Holzminden, Germany
Imre Blank Nestle
´
Research Center, Lausanne, Switzerland
Stefan Brennecke Haarmann & Reimer GmbH, Holzminden, Germany
Wilfried Bretschneider Haarmann & Reimer GmbH, Holzminden, Germany
Alain Chaintreau Firmenich S.A., Geneva, Switzerland
Elaine T. Champagne Southern Regional Research Center, Agricultural Re-
search Service, U.S. Department of Agriculture, New Orleans, Louisiana
Ben D. Gardner Orion Associates Science & Engineering, Okemos, Michigan
Casey C. Grimm Southern Regional Research Center, Agricultural Research
Service, U.S. Department of Agriculture, New Orleans, Louisiana
Alan D. Harmon Research and Development, McCormick & Co., Inc., Hunt
Valley, Maryland
John F. Holland Department of Biochemistry, Michigan State University, East
Lansing, Michigan
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
John Labows Colgate-Palmolive Company, Piscataway, New Jersey
Steven W. Lloyd Southern Regional Research Center, Agricultural Research
Service, U.S. Department of Agriculture, New Orleans, Louisiana
Ray Marsili Dean Foods Company, Rockford, Illinois
Thomas McGee Global Technology and Innovation, Givaudan Fragrances
Corp., Teaneck, New Jersey
Robert J. McGorrin Department of Food Science and Technology, Oregon
State University, Corvallis, Oregon
James A. Miller Southern Regional Research Center, Agricultural Research

Service, U.S. Department of Agriculture, New Orleans, Louisiana
Ken’ichi Ohtsubo National Food Research Institute, Ministry of Agriculture,
Forestry and Fisheries, Ibaraki-Ken, Japan
Thomas Parliment Parliment Consulting, New City, New York
Richard Payne Colgate-Palmolive Company, Piscataway, New Jersey
Allen E. Puchalski Colgate-Palmolive Company, Piscataway, New Jersey
Kenneth L. Purzycki Givaudan Fragrances Corp., Teaneck, New Jersey
Arthur M. Spanier Beltsville Agricultural Research Center, Agricultural Re-
search Service, U.S. Department of Agriculture, Beltsville, Maryland
Thomas P. Wampler CDS Analytical, Inc., Oxford, Pennsylvania
Peter Werkhoff Haarmann & Reimer GmbH, Holzminden, Germany
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
1
Solvent Extraction and Distillation
Techniques
Thomas Parliment
Parliment Consulting, New City, New York
I. INTRODUCTION
The purpose of this chapter is to review techniques that have been published
in the technical literature and developed in our laboratory for the isolation and
concentration of samples prior to analysis by gas chromatography. It is our goal
to emphasize those techniques that are easy to employ, require minimal equip-
ment, and produce reproducible, meaningful results. In a number of cases, exam-
ples of the results will be presented.
As has been described previously (1), sample preparation is complicated
by a number of factors:
1. Concentration Level: Aromatics levels are generally low, typically in
the ppm, ppb, or ppt range. Thus, it is necessary not only to isolate
the components but also to concentrate them by several orders of mag-
nitude.

2. Matrix: The volatiles are frequently intracellular and must be liberated
by disruption. The sample frequently contains nonvolatile components
such as lipids, proteins, or carbohydrates, which complicates the isola-
tion process. These components may create problems of foaming and
emulsification during isolation procedures and will create artifacts if
injected into a hot gas chromatography injector port.
3. Complexities of Aromas: The aromatic composition of foods are fre-
quently very complex. For example, coffee currently has almost 800
identified components, as shown in Table 1. Complicating the picture
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
T
ABLE
1 Classes of Aroma Compounds in Coffee
Number of
Chemical class compounds
Hydrocarbons 74
Alcohols 20
Aldehydes 30
Ketones 73
Acids 25
Esters 31
Lactones 3
Phenols (and ethers) 48
Furans 127
Thiophenes 26
Pyrroles 71
Oxazoles 35
Thiazoles 27
Pyridines 19
Pyrazines 86

Amines and miscellaneous nitrogen compounds 32
Sulfur compounds 47
Miscellaneous 17
Total 791
Source: Ref. 2.
is the fact that the classes of compounds present cover the range of
polarities, solubilities, and pHs.
4. Variation of Volatility: The components possess boiling points ranging
from well below room temperature to those that are solids, such as
vanillin (mp 81°C).
5. Instability: Many components in an aroma are unstable and may be
oxidized by air or degraded by heat or extremes of pH.
Regardless of which sample preparation technique is employed, it is criti-
cally important to assess the organoleptic quality of the isolate. No single tech-
nique will prove optimal for every sample, and evaluations should be made to
ensure that decomposition and loss of desired components do not occur. A very
significant paper published by Jenings et al. (3) compared various sample prepara-
tion techniques, including porous polymer trapping and distillation-extraction.
Their conclusion was that no isolation technique produced results that duplicated
the original neat sample, but that distillation-extraction most nearly agreed
(Fig. 1).
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
F
IGURE
1 Relative integrator response for various sample preparation techniques. (From
Ref. 3.)
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
This is particularly important since current flavor research seems to be less
directed to identification for the sake of adding to the numbers of the compounds
in the knowledge base, and more to alternative reasons. At the present time it

appears one purpose is characterization of components of organoleptic impor-
tance. Three techniques for gas chromatographic individual component assess-
ment are in vogue: aroma extraction dilution analysis (AEDA), calculation of
odor units, and CharmAnalysis (see Chapter 12). Another purpose of flavor re-
search is to analyze products and to perform flavor stability studies.
At the present time, the two most common procedures reported in the litera-
ture for the isolation of the aromatics are headspace methods and extraction. The
former will be covered in the next chapter. The purpose of this chapter is to
review techniques for isolating and concentrating aromatics, which include vari-
ous distillation and extraction procedures.
A number of references exist on the topic of flavor isolation, and these
provide a different perspective on the topic (4–8). To quote Schreier (9): ‘‘It
must be emphasized that sample preparation is the most critical step in the entire
analytical process of the investigation of volatiles.’’
II. DIRECT INJECTION OF THE SAMPLE
A. Essential Oils
Direct injection is by far the most convenient technique and works particularly
well for essential oils. The sample may have to be diluted with a solvent to obtain
response within the limits of the detector.
B. Aqueous Samples
When concentrated aqueous samples are available, direct injection techniques
can be employed. In industry, aqueous materials are frequently available from
industrial operations. Examples of this would be condensates from coffee grind-
ers, vapors from chocolate conching operations, and aqueous materials from cit-
rus juice concentrators.
The aqueous phase may be injected if the sample is sufficiently concen-
trated. A number of problems may be encountered under these circumstances.
When water is converted to steam, the volume increases dramatically; 1 µlof
water becomes more than 1000 µl of steam. This is larger than the injector volume
of many current gas chromatographs, and the steam may degrade the performance

of the system. Polar gas chromatography liquid phases such as Carbowax and
PEG will degrade in the presence of steam unless they are bonded to the column.
If the aqueous sample contains dissolved solutes such as carbohydrates or
proteins, additional problems will arise when the sample is injected. The nonvola-
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
tiles may decompose, leaving a nonvolatile residue in the injector and at the head
of the column. Many researchers use a guard column of deactivated fused silica
tubing between the injector and the analytical column. The guard column can be
replaced periodically when it becomes contaminated. The tubing contains no liq-
uid phase, thus it does not affect separation or retention time. The guard column
can be connected to the analytical column with various types of press-tight con-
nectors (10).
If the aqueous phase is too dilute, concentration techniques as described
in the next section may be employed.
III. DIRECT SOLVENT EXTRACTION OF AQUEOUS
SAMPLES
Aqueous samples are available from a number of sources. Industrial plant opera-
tions may yield such products. Carbonated beverages, fruit juices, and caffeinated
beverages can often be extracted directly. Fruits and vegetables can be homoge-
nized with water, treated with a pectinase enzyme to destroy the pectins, and
filtered through a bed of diatomaceous earth to remove particulates.
A. Extraction
When relatively large amounts of aqueous samples are available, then separatory
funnels or commercial liquid-liquid extractors may be employed. A large number
of solvents have been summarized by Weurman (4) and reviewed by Teranishi
et al. (5).
The solvents most commonly used today are diethyl ether, diethyl ether/
pentane mixtures, hydrocarbons, Freons, and methylene chloride. The latter two
have the advantage of being nonflammable. Solvent selection is an important
factor to consider, and the current status has been summarized by Leahy and

Reineccius (11). In general, the following suggestions can be made. Nonpolar
solvents such as Freons and hydrocarbons should be used when the sample con-
tains alcohol. Diethyl ether and methylene chloride are good general purpose
solvents. Ether can form explosive peroxides, and for that reason contains inhibi-
tors (e.g., BHT), which will show up in gas chromatography/mass spectroscopy
(GC/MS) analysis. We find that methylene chloride is a satisfactory general pur-
pose solvent, particularly for flavor compounds with an enolone structure (e.g.,
Maltol and Furaneol). It is somewhat toxic and is an animal carcinogen. To aid
in extraction, sodium chloride may be added to the aqueous phase to salt out the
organics when low-density solvents are employed.
If the sample contains any particulates, it should be filtered. A convenient
way to filter samples is through a syringe filter (e.g., Gelman Sciences, Ann
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
Arbor, Mich.) of the type recommended for HPLC sample preparation. These
filters have a pore size of 0.45 µm and are solvent resistant. Microtypes with low
solvent hold-up are available.
Figure 2 shows the total ion chromatogram of a coffee extract. In this case
a decaffeinated roast and ground coffee was brewed in a commercial system.
The brew was filtered through a Gelman 0.45 µm GHP Acrodisc to remove partic-
ulates, and the aqueous phase was extracted with methylene chloride. A highly
complex chromatogram is evident. The large peak eluting at 25 minutes is caf-
feine.
Continuous extractors have been described in the literature for solvents
more dense and less dense than water (e.g., Ref. 4) and are available commer-
cially (e.g., ACE Glass, Vineland, NJ; Supelco, Inc., Bellefonte, Pa) for $200–
600 (Fig. 3). These are a pleasure to use (providing there is no solvent loss and
that emulsions don’t occur) since they will operate relatively unattended. They
are normally operated for 2–4 hours, but may be operated overnight.
Liquid carbon dioxide was recommended as an extracton solvent as early
as 1970 (12). It has the advantages of being nontoxic and inexpensive. Liquid

carbon dioxide is reported to have solvent properties similar to diethyl ether (12)
and to be particularly selective for esters, aldehydes, ketones, and alcohols. If
water is present, it will be removed also.
A commercial liquid carbon dioxide Soxhlet extractor is commercially
available (J&W Scientific, Folsom, CA). The vessel holds a sample of 2.5 g.
F
IGURE
2 Total ion chromatogram (TIC) of brewed R&G coffee extracted with methy-
lene chloride.
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
F
IGURE
3 Liquid/liquid extractor concentrator apparatus. (Courtesy Supelco, Inc.,
Bellefonte, PA.)
This apparatus seems to have achieved only limited use, perhaps because of its
cost ($1500 plus accessories) and limited sample size. Moyler (13) discussed a
commercial liquid carbon dioxide system and reported such extracts to be more
concentrated than the steam distillates or solvent extracts. More important, he
reported that the character was ‘‘finer.’’
Supercritical carbon dioxide has been employed recently as an extraction
solvent. When using supercritical carbon dioxide, it is necessary to balance tem-
perature, pressure, and flow rate, which requires complex instrumentation. Sev-
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
eral instrument vendors produce supercritical fluid extractors in the price range
of $25,000–90,000. Again, sample capacity is relatively limited.
B. Emulsions
Emulsions can be a problem, particularly if nonvolatile solutes are present. To
prevent emulsions, the following methods can be employed:
Use gentle shaking.
Filter the sample if particulates are present.

Keep the system cool.
Be patient.
Adjust the pH of the aqueous phase.
The latter technique is particularly effective if organic acid, basic, or amphoteric
compounds are present. If emulsions occur, centrifugation may be employed (but
only for nonflammable solvents).
C. Concentration
The final step is concentration of the solvent. We usually dry the solvent over
sodium sulfate or magnesium sulfate and then carefully concentrate it on a steam
bath using a Vigreux column. A convenient method to concentrate large volumes
of solvent is by use of a Kuderna-Danish Evaporative Concentrator, which is
available in both macro (up to 1000 ml) and micro (1–4 ml) capacities for less
than $100.
D. Impurities
High-boiling impurities both in solvent and sample will also be concentrated
along with the desired analytes. Thus, solvent blanks should be prepared. If the
sample was a direct extract, the solvent will contain nonvolatile components such
as natural and Maillard pigments, lipids, alkaloids, etc. These may crystallize or
precipitate on concentration and will leave a residue in the injector of the gas
chromatograph.
For additional suggestions on extracting aqueous samples, see Secs. IV
and V.
IV. STEAM DISTILLATION OF SAMPLES FOLLOWED
BY SOLVENT EXTRACTION
One of the most common sample-preparation techniques employed today in-
volves steam distillation followed by solvent extraction. The primary advantage
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.