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Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R.. Handbook of Fruit Science and Technology: Production, Composition, Storage, and Processing, edited by D.. Handboo

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Techniques for Analyzing Food Aroma

Food Science and Technology

A Series of Monographs, Textbooks, and Reference Books

EDITORIAL BOARD

Owen R Fennema University of Wisconsin – Madison

Marcus Karel Rutgers University Gary W Sanderson Universal Foods Corporation Steven R Tannenbaum Massachusetts Institute of Technology Pieter Walstra Wageningen Agricultural University John R Whitaker University of California – Davis

1 Flavor Research: Principles and Techniques, R Teranishi, I Hornstein, 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

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15 Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R Fennema

16 Sensory Evaluation of Food: Statistical Methods and Procedures, Michael O'Mahony

17 Alternative Sweetners, 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, edited 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

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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 Perspectives, 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

Additional Volumes in Preparation

Food Proteins and Their Applications, edited by Srinivasan Damodaran and A Paraf

Techniques for Analyzing Food Aroma

Edited By Ray Marsili

Dean Foods Company Rockford, Illinois

MARCEL DEKKER, INC

NEW YORK • BASEL

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Library of Congress Cataloging-in-Publication Data

Techniques for analyzing food aroma / edited by Ray Marsili

p cm.— (Food science and technology ; 79)

Includes index

ISBN 0-8247-9788-4 (hardcover : alk paper)

1 Food—Sensory evaluation I Marsili, Ray

II Series: Food science and technology (Marcel Dekker, Inc.) ; 79

TX546.T43 1997

664'.07—dc20 96–36593

CIP

The publisher offers discounts on this book when ordered in bulk quantities For more

information, write to Special Sales/Professional Marketing at the address below

This book is printed on acid-free paper

Copyright © 1997 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 record-

ing, or by any information storage and retrieval system, without permission in writing

from the publisher

Marcel Dekker, Inc

270 Madison Avenue, New York, New York 10016

Current printing (last digit):

10 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

To Laura, Amy Nathan, and Jason

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The search for Truth is in one way hard and in another way easy For it is evident that no one can master it fully nor miss it completely But each adds a little to our knowledge of Nature, and from all the facts assembled there arises a certain grandeur

Aristotle

Preface

Flavor is of major concern to food scientists because it is a significant factor influencing the public's food-buying decisions and its perception of food quality Analyzing the volatile and semivolatile organic compounds that impact the flavor and aroma of foods can be a daunting task, and obtaining useful information from such measurements can be even more challenging

It is the intention of the authors to describe analytical techniques that can be applied to the detection and quantitation of volatile aroma chemicals in foods and to explain how the sense of smell

(olfactory detection) can be incorporated with these techniques to resolve important, practical

problems related to food aromas Advantages, disadvantages, and biases of each technique are discussed, as well as when and why specific techniques should be selected or avoided

The chapters contain dozens of examples of applications showing how real food aroma problems have been resolved through the use of modern analytical instruments and olfactometry Specifically, the book discusses various sample preparation techniques for isolating and concentrating food aroma compounds prior to gas chromatographic (GC) analysis; how GC column technology, column manipulation techniques, and GC/MS detection can be used to maximize resolution, discrimination, identification, and sensitivity for detecting important aroma-influencing components; and how sensory techniques, including the use of an olfactometry detector, can be combined with

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chromatography (SFC), and other analytical methods, GC techniques are emphasized in this work since they are generally more applicable to the analysis of volatile organic polar compounds, which are frequently the most important contributors to food aroma.

The dedication, persistence, and splendid cooperation of all contributing authors, as well as the quality of information they have presented, are to be commended Also, I would like to

acknowledge my indebtedness to my associates, Gregory J Kilmer, Nadine Miller, and Ronald E Simmons, and to my supervisor, Dr Scott Rambo, for their advice, encouragement, and continual support Special thanks go to my wife, Deborah, for reviewing the chapters and for her patience and words of encouragement

RAY MARSILI

Alexander Bernreuther, Ulrich Epperlein, and Bernhard Koppenhoefer

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Behroze S Mistry, Terry Reineccius, and Linda K Olson

9 Gas Chromatography-Olfactometry for the Determination of Key

Contributors

Alexander Bernreuther Joint Research Center, Environment Institute, Ispra, Italy

Imre Blank Research Centre, Nestec Ltd., Lausanne, Switzerland

Ulrich Epperlein University of Tübingen, Tübingen, Germany

Casey C Grimm Southern Regional Research Center, Agricultural Research Service, United States

Department of Agriculture, New Orleans, Louisiana

Alan D Harmon Research and Technical Development, McCormick & Co., Inc., Hunt Valley,

Maryland

Diana Hodgins Wheathampstead, Hertfordshire, England

Charles K Huston Varian Chromatography Systems, Walnut Creek, California

Berhard Koppenhoefer Institut für Organische Chemie der Universität, University of Tübingen,

Tübingen, Germany

Steven W Lloyd Southern Regional Research Center, Agricultural Research Service, United States

Department of Agriculture, New Orleans, Louisiana

Ray Marsili Dean Foods Company, Rockford, Illinois

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James A Miller Southern Regional Research Center, Agricultural Research Service, United States

Department of Agriculture, New Orleans, Louisiana

Behroze S Mistry Aspen Research Corporation, St Paul, Minnesota

Linda K Olson Aspen Research Corporation, St Paul, Minnesota

Thomas H Parliment Kraft Foods, White Plains, New York

Terry Reineccius Aspen Research Corporation, St Paul, Minnesota

Arthur M Spanier Southern Regional Research Center, Agricultural Research Service, United

States Department of Agriculture, New Orleans, Louisiana

Thomas P Wampler CDS Analytical, Inc., Oxford, Pennsylvania

Donald W Wright Microanalytics Instrumentation Corporation, Round Rock, Texas

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 equipment, and produce reproducible, meaningful results In a number of cases, examples 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 not only necessary to isolate the components, but also to concentrate them by several orders of magnitude

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Page 2

Table 1 Classes of Aroma Compounds in Coffee

5 Instability: Many components in an aroma are unstable and may be oxidized by air or degraded

by heat or extremes of pH

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Regardless of which sample preparation technique is employed, it is critically important to assess the organoleptic quality of the isolate No single technique 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 Jennings et al (3) compared various sample

preparation 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)

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 importance Three techniques for gas chromatographic individual component

assessment are in vogue: aroma extraction dilution analysis (AEDA), calculation of odor units, and Charm Analysis (see Chapter 9) Another purpose of flavor research is to analyze products and to perform flavor stability studies

At the present time, the two most common procedures reported in the literature 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 various 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

Figure 1 Relative integrator response for various sample preparation

techniques (From Ref 3.)

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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 nonvolatiles 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 liquid 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 connectors (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 operations may yield such products Carbonated beverages, fruit juices, and caffeinated beverages can often be extracted directly Fruits and vegetables can be homogenized 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 contains alcohol Diethyl ether and methylene chloride are good general purpose solvents Ether can form explosive peroxides, and for that reason contains inhibitors (e.g., BHT), which will show up in gas chromatography/mass spectroscopy (GC/MS) analysis We find that methylene chloride is a satisfactory general purpose 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 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 particulates, and the aqueous phase was extracted with methylene chloride A highly complex chromatogram is evident The large peak eluting at 25 minutes is

caffeine

Continuous extractors have been described in the literature for solvents more dense and less dense than water (e.g., Ref 4) and are available commercially (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 This apparatus seems to have achieved only limited use, perhaps because of its cost ($1500 plus accessories) and limited sample size Moyler (13) dis-

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Figure 2 Total ion chromatogram (TIC) of brewed R&G coffee extracted

with methylene chloridecussed 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 temperature, pressure, and flow rate, which requires complex instrumentation Several 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

Figure 3 Liquid/liquid extractor concentrator apparatus (Courtesy Supelco, Inc, Bellefonte, PA.)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

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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 Sections IV and V

IV Steam Distillation of Samples Followed by Solvent Extraction.

One of the most common sample-preparation techniques employed today involves steam distillation followed by solvent extraction The primary advantage is that the distillation step separates the volatiles from the nonvolatiles Other reasons for this include simplicity of operation, no need for complex apparatus, reproducibility, rapidity, and the range of samples that can be handled Steam distillation works best for compounds that are slightly volatile and water insoluble In addition, compounds with boiling points of less than 100°C will also pass over

A Direct Distillation

The sample is normally placed in a round-bottom flask and dispersed in water The aqueous slurry can be heated directly (with continuous stirring) to carry over the steam-distillable components Problems can be encountered due to scorching of the sample if too much heat is applied, and in addition bumping may occur when the sample contains particulates Stirring may prevent these problems Foaming is another potential problem Many food products contain surface-active agents and will foam during distillation; addition of antifoams (e.g., DC polydimethyl siloxanes) may prevent this problem, but these silicones usually end up in the distillate, as evidenced by GC-MS peaks at m/z = 73, 147, 207, 221, 281, and 341

B Indirect Steam Distillation

Indirect steam distillation has many advantages over the direct technique It is more rapid and less decomposition of the sample occurs since the sample is not heated directly The steam may be generated in an external electrically heated steam generator or in a round-bottom flask heated by a mantle It is even possible to use laboratory house steam, in which case the steam must be passed through a trap that allows removal of condensate and any particulates that may come out of the line

It is imperative that blank samples be run, since house steam may be highly contaminated Even so, this technique has the great advantage of being rapid and easy The steam and volatiles are usually condensed in a series of traps cooled with a succession of coolants ranging from ice water to dry ice/acetone or methanol

C Vacuum Steam Distillation

It sample decomposition remains a concern, then the steam distillation may be operated under vacuum In this case inert gas should be bled into the system to aid in agitation A number of cooled traps should be in line to protect the pump from water vapor and the sample from pump oil vapors Another simple method to generate a condensate under vacuum is by use of a rotary evaporator Bumping is normally not a problem in this case The higher-boiling components do not distill as efficiently as they do under atmospheric pressure

Once the vapors have been condensed, it remains to extract the sample, which is normally very dilute Techniques described in Section III may be employed In addition, there are two semi-micro extraction techniques that have value

D Extraction

Use of the Mixxor has been described by Parliment (14) and its utility described in a number of publications (15,16) Such a device is shown in Figure 4 These extractors are available with sample volumes ranging from 2ml to 100ml The 10-ml capacity extractor is particularly convenient

capacity for flavor research Briefly, approximately 8ml of aqueous condensate is placed in receiver

B and saturated with sodium chloride The whole assembly is cooled and then a quantity of diethyl ether (typically 0.5–0.8ml) is added The ether may contain an internal standard The system is extracted by moving chamber A up and down a number of times After phase separation occurs, the solvent D is forced into an axial chamber C, where it can be removed with a syringe

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Figure 4 Mixxor apparatus for the extraction of aqueous samples (From Ref 14.)for analysis Percent recoveries for a series of ethyl esters from an aqueous solution were essentially quantitative even at the sub-ppm level Unfortunately, these extractors were imported from Europe, and they are becoming difficult to procure

A less sophisticated alternative exists The sample may be placed in a screw-capped centrifuge tube and a small amount of dense solvent added After exhaustive shaking, the tube can be centrifuged to break the emulsion and separate the layers The organic phase can be sampled from the bottom of the tube with a syringe Methylene chloride works well in this application

Figure 5 compares the total ion chromatograms of two samples Roasted and ground coffee was indirectly steam distilled at atmospheric pressure and a condensate collected The upper curve in the figure represents the ethereal concentrate prepared via the Mixxor technique; the lower curve is the methylene chloride extract Pattern differences are apparent The largest peak in the ethereal extract (Rt = 6.0) is furfuryl alcohol; the largest peak in the lower curve (Rt = 8.5) is 5-methyl furfural

Figure 5 Comparison of chromatograms of etheral (upper) and methyl-

ene chloride (lower) extract of R&G coffee

E Manipulation of the Aqueous Phase.

Adjustment of the pH of the aqueous phase before extraction may accomplish two goals First, emulsions may be broken, permitting phase separation to take place rapidly Second, class

separation will take place, which may simplify the gas chromatographic pattern This is less

necessary today since contemporary gas chromatography columns have high resolving power; however, frequently

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Figure 6 shows an example of such a manipulation In this case the upper curve is the ethereal extract of a steam distillate of coffee at pH 3.6 The larger asymmetrical peaks at Rt = 5.8 and 6.8 represent fatty acids These are eliminated at pH 10.0 (middle curve) The large peak at Rt = 3.8 in the latter is pyridine, a decomposition product of trigonilline The lower curve in this figure is the material that remains after bisulfite extraction It is immediately apparent that many of the lower boiling components of coffee are carbonyl in nature For example, the peak at Rt = 5.5 is furfural In this manner it is possible to simplify the gas chromatographic pattern.

If the aqueous phase is limited in quantity, the analyst can perform an interesting set of sequential experiments The sample is placed in the Mixxor Chamber B, the pH adjusted to about 3 with acid, and the sample extracted with diethyl ether Sufficient sample is removed for gas chromatographic analysis, e.g., 1 μl The aqueous phase is made alkaline and the sample reextracted with the same diethyl ether and gas chromatographic analysis repeated Finally, the sample is made neutral and saturated with sodium bisulfite and reextracted The ethereal phase is reanalyzed In this case three different analyses can be made from the same sample in a short period of time and subjected to GC-

MS and organoleptic analysis

V Simultaneous Steam Distillation/Extraction

One of the most popular and valuable techniques in the flavor analysis field is the simultaneous steam distillation/extraction (SDE) apparatus first described by Likens and Nickerson (17) The apparatus provides for the simultaneous condensation of the steam distillate and an immiscible organic solvent Both liquids are continuously recycled, and thus the steam distillable-solvent soluble compounds are transferred from the aqueous phase to the solvent The advantages of this system include the following:

1 A single operation removes the volatile aromas and concentrates them

2 A small volume of solvent is required, reducing problems of artifact buildup as solvents are concentrated

Figure 6 Comparison of chromatograms of R&G coffee extracted at pH 3.6 (upper curve), pH 10.0 (middle curve), and with bisulfite (lower curve).

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3 Recoveries of aroma compounds are generally high

4 The system may be operated under reduced pressure to reduce thermal decomposition

A number of refinements have been made to the basic apparatus, some of which are shown in Figure 7a-d (9) One version is commercially available for less than $300 (J&W Scientific, Folsom, Ca); a diagram of it is shown in Figure 7e

Typically the sample flask has a 500 ml to 5 liter capacity and contains the sample dissolved or dispersed in water so that the flask is less than half filled Agitation is advisable if suspended

materials are present to prevent bumping As with all distillations, the pH of the sample should be recorded (and adjusted if necessary) prior to distillation Heat may be supplied by a heating mantle

or (better if solids are present) a heated oil bath with stirrer The solvent is normally contained in a pear-shaped flask of 10–50ml capacity Many solvents have been employed In one model system study, Schultz et al (18) compared various solvents as the extractant They reported that hexane was

an excellent solvent except for lower-boiling water-soluble compounds, where diethyl ether was considerably better Use of methylene chloride has been recommended in a modified Likens-

Nickerson extractor (19) Currently, most researchers appear to be using pentane-diethyl ether mixtures

Regardless of which solvents are used, boiling chips should be added to both flasks to ensure

smooth boiling The distillation is generally performed for 1–3 hours After the distillation is

completed, the system is cooled and the solvent from the central extracting U tube is combined with that of the solvent flask The solvent is dried over an agent such as sodium sulfate and concentrated

by slow distillation

An impressive example of the use of a Likens-Nickerson extractor is shown in Figure 8 This figure shows the gas chromatogram of a green and a roasted Kenyan coffee and shows how aromatic compounds are generated in the roasting process (W Holscher, personal communication)

Vacuum versions of the SDE system have been described These have the advantage of reducing the thermal decomposition of the analyte Leahy and Reineccius report (11) that vacuum operation had

a slightly negative effect upon recovery compared to atmospheric operation Our experience is that operation under vacuum is quite complex since one must balance the boiling of two flasks, keep the solvent from evaporating, and hold the pressure constant

Table 2 presents results of a series of experiments wherein typical flavor compounds in a model mixture were isolated by various SDE techniques In

Figure 7 Various modificatins to SDE apparatus (a,b,c,d from Ref 9; e Courtesy J&W Scientific, Folsom, CA.)

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Figure 7 Continuedgeneral, ether is a better solvent than hydrocarbons, and atmospheric pressure better than reduced pressure

VI Direct Solvent Extraction of Solid Samples

An entirely different process of sample-preparation technique involves direct solvent extraction, which is a very simple and convenient technique Probably the easiest way to do such an extraction

is with a Soxhlet extractor A dried sample such as a spice, chocolate nib, R&G coffee, or a grain can be ground finely and placed in a Soxhlet thimble and extracted with an organic solvent Either diethyl ether or methylene chloride may be used in such a system After a number

Figure 8 Chromatographic comparison of green and roasted coffee (W

Holscher, personal communication.)

of cycles, the solvent can be combined and concentrated Nonvolatile organic materials such as lipids, alkaloids such as caffeine and theobromine, and pigments will also be concentrated The sample may be analyzed directly (with trepidation) or it may be treated as described in the section below, after removal of the solvent If the sample contained large amounts of lipids (e.g., coffee, chocolate), then the volatiles may be removed by subsequent steam distillation or by a high vacuum stripping technique as described in Section VII

Figure 9 is the GC-MS of a roast and ground coffee sample, which was moistened with water and extracted with methylene chloride in a Soxhlet extractor The large component eluting at 26 minutes

is caffeine

VII High Vacuum Distillation of Lipids

A number of the procedures described in Section VI will yield a material that is primarily lipid in nature In addition, many samples available to the researcher are themselves lipids A few materials that one may encounter are coffee oil, vegetable and nut oils, cocoa butter, lard, butter oil, lipids used for deep fat frying, and lipids used as the solvent for Maillard reaction systems

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Table 2 Recovery of Components by SDE from the Model Mixture at a Concentration of 165 ppm (w/v) for Each Compound

(Recovery as Percentage of Initial Amount)

1.0 ml of glacial acetic acid, titrated in solution to pH 5 with sodium hydroxide, was also present in this run in addition to the

usual citrate buffer at 0.05 M.

Source: Ref 18.

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Figure 9 TIC of a roast and ground coffee sample moistened with water

and extracted with methylene chloride.

Such materials can be a relatively rich source of aromatic compounds since aroma compounds are typically lipid soluble A number of procedures can be used to prepare a sample In this section we will cover three useful ones

A Steam Distillation

The lipid material may be steam distilled at atmospheric pressure or under vacuum, as was

described in Section IV, and subsequently subjected to solvent extraction Alternatively, a modified Likens-Nickerson extractor has been described (19), which permits the introduction of steam into the system Recoveries of model compounds from lipid systems were not as satisfactory as for aqueous samples

B High Vacuum Distillation.

When large amounts of lipid materials are present, the sample may be subjected to a falling film molecular still The apparatus utilizes the principle of vaporization of the flavor from a heated thin film of the oil under high vacuum One such apparatus is shown in Figure 10 (20) Several hundred milli-

Figure 10 Falling film molecular still for the removal

of volatiles from lipids (From Ref 20.)liters of oil are placed in vessel A and slowly passed through the foaming chamber into the heated bellows chamber The distillate is collected in a series of traps cooled with liquid nitrogen The oil may be recycled Another series of apparatus described by Chang et al at Rutgers (21) has

accomplished similar goals This type of apparatus generally falls into the same category of

equipment as that used to deodorize lipids

C Short Path Distillation

One version of the apparatus is shown in Figure 11a The nonvolatile material is placed in the flask The flask is heated while stirring the sample and a high vacuum is applied The inner condenser is cooled with liquid nitrogen or dry ice-solvent (22) We have found this apparatus very useful for separating the volatile aromatics from nonvolatile residues (i.e., lipids) such as those gener-

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Figure 11 Apparatus for the removal

of aromatics from lipids (a from Ref 22;

b from Ref 23.)ated in Section VI In that case the sample size may be only a few grams or less, and a smaller version of the short path distillation apparatus is appropriate This apparatus can be easily fabricated

by a glassblower

An example of the application of such an apparatus is shown in Figure 12 The sample was

produced by high vaccum distillation of 10 g of coffee oil

ex-Figure 12 TIC of volatiles from roast and ground coffee oil, distilled in

apparatus shown in Figure 11a.