Report
GC/OLFACTOMETRY
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wenty years before the development of the gas chromatograph, scientists studying human olfaction built instruments to deliver controlled doses of odorants to human subjects for two purposes: to study the human olfactory process and to quantify the sensory activity of chemicals (1,2). Soon after publication of the seminal paper on GC in 1952 researchers concerned with flavor and fragrance began sniffing GC effluents to detect odor-active chemicals (odorants) separated by GC. Because they were interested only in chemicals with odor the GC with its superior separation small sample capacity and ability to deliver analytes in a gas phase ready to be sniffed was a revolutionary technology Researchers could detect chemicals at the CC that were not detectable by any other rhemical detectors and to some extent this is still true todav Taken tnoerher these instruments and protnrols are called
olfactometry. In 1964, Fuller and colleagues at the Colgate-Palmolive Co. published a paper complete with photographs that described a system for sniffing GC effluents (3). Called the "perfume detector", their system incorporated the essential components commonly used by most researchers at that time to sniff GC effluents: : GC Cith a chemical detector followed by or in parallel with a "sniff port" and a trained human sniffer. The special booth used in the Colgate system to isolate and ventilate the sniffer exemplifies the central problem with the technique standardizing and quantifying the response given by the sniffer
GC combined with olfactometry can beusedto study the human olfactory process and to quantify the sensory activityof chemicals
Over the next 30 years, sniff ports began to incorporate design features found in gas-phase olfactometers, while sniffer
Terry E. Acree Cornell University 170 A
Analytical Chemistry News & Features, March 1, 1997
0003-2700/97/0369-170A/$14.00/0 © 1997 American Chemical Society
GC WITH A SENSE OF SMELL training and data handling methods began to include some of the practices commonly used in sensory testing. Together, these methods are called GC/olfactometry (GC/O). GC/O is as important in the analytical chemistry of flavor and fragrance materials as GC/MS, despite GC/O's greater noise and bias and higher cost; the specific sensitivity of GC/O for odorants often exceeds the sensitivity of GC/MS due to the ingenious design of the human olfactory system. The olfactory systems of organisms consist of transduction, a process for converting a chemical pattern into electrochemical impulses that are transmitted to the brain, and pattern recognition programs that the brain uses to convert these impulses into precepts that mediate behavior. Transduction occurs on the tissue surface at the top of the nasal cavity, called the olfactory epithelium. Recent studies have confirmed that the receptor sites on the olfactory epithelium are proteins that loop in and out of the receptor cell membrane times a characteristic called "seven transmembrane G-protein counted receptors" These olfactory receptor proteins are extremely sensitive, and selective systems amplify signals in much the same way that photomultipliers amplify electrons— through amplifying cascades. These receptor proteins have a primordial design and share their structural features with different species. This similarity implies that different species may "smell" the same or similar chemicals, similar to the way that different species respond to the same wavelengths of light. Thus many organisms may have similar transduction systems that differ mainly in their patternrecognition programs Therefore it is not surprising that GC/O has been used to identify insect pheromones and weasel
scent-gland components (4 Ti This arti-
cle will summarize the technical development, application, and potential use of GC/O to measure the odor of materials. From sniff port to dynamic olfactometry
The simplest and probably the earliest method for sniffing GC effluents was to sit close to the exit port of a nondestructive detector, such as a thermal conductivity detector, or to stand over the exhaust of a flame ionization detector (with the hydrogen gas turned off, of course) placing your nose close to the detector exit and sniffing the hot exhaust gases (3). Because the GC was not designed as an olfactometer, the sniffer was often uncomfortable frequently disturbed by the dehydrating effects of the gases and sometimes burned by the hot metal surfaces of the detectors. A strong background of odorants emitted by hot insulation and plastic components interfered with the detection of odorants eliitinp" from the GC and added to the freneral discomfort associated with the proceHnrp lVtnst lahnratories that nAPrlprt to use extensive GC sniffim? addressed nroblems with design modifications in
eluding heated transfer lines to move the exit port away from the GC equipment, ventilated environments to clear effluents r
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from the sniffer, and various masks and ports designed to create a more comfortable environment. Two general types of olfactometers are used: static and dynamic. Static olfactometers can be as simple as plastic squeeze bottles filled with solutions of odorants in an odorless carrier, usually water. Because of their simplicity, portability, and standardizability, they are still the most frequently used olfactometers for research and consumer testing. Static systems always deliver to the subject a pulse of odorant mixed in humid air (~ 100% RH) whercfls the dyriciffiic systems CBH deliver
a constant dose in a background of lesshumid air. Because adaptation, or the loss of sensitivity with exposure to odorant, is a central feature of most olfactory systems, dynamic olfactometers are generally pulsed with square-wave odorant profiles in a background of constant humidity (often ~ 50% RH). In both methods, some diligence is required to know the exact composition of any given dose, because volatilities, flow rates, and inspiration patterns can affect the response of a subject to any given dose, and measuring these is not always practical or even possible Nevertheless excellent and illuminating results have been obtained with both tvoes of olfactometers (6) In 1971, Dravnieks and O'Donnell at the Illinois Institute of Technology applied their understanding of olfactometry to the GC sniff port and developed a true GC/O (7). Central to their design was the dilution of the hot GC effluent gases with humidified air, which resulted in a dynamic olfactometer delivering to the sniffer a pulsed stimulant dose with a Gaussian distribution concentration gradient and a bandwidth similar or slightly larger than that generated by the GC column. The improvement over existing sniff port technology was immediately obvious. Performance was subsequently enhanced by a venturi
Figure 1 . GC/O with the sniffer seated in front of a terminal recording odor perceptions.
Analytical Chemistry News & Features, March 1, 1997 171 A
Report tering an odorant, the odor character is so unique that only a few chemical structures will produce the same exact response. This leads to a useful maxim: if two chemicals have the same retention time and the same odor character, they are the same chemical. If one of the chemicals is an authentic standard, the other has been identified with considerable certainty. The obvious exception to this maxim would be the GC/O of diastereomers with the same odor which is not a However the of chiral-specific GC columns when the standards show chiral features would remove any remaining doubt For those chemicals with odor GC/O is one of the most Dowerful authenticating terhninnes svailahle en aanlvtiraa
chemists assuming Figure 2. T h e p r o c e s s f o r c o l l e c t i n g a n d r e p o r t i n g GC/O d a t a b a s e d o n t h e t w o m o s t c o m m o n s e r i a l d i l u t i o n p r o t o c o l s , AEDA a n d C h a r m A n a l y s i s .
system using inert make-up gases and controlled humidity, resulting in a dynamic GC/Ofreeof background odors, delivery to the sniffer in a comfortable position (Figure 1), and the capability of handling narrow-bore (0.3-mm i.d.) capillary columns without serious loss in resolution (8). Evolution in the design of the GC/O to preserve laminar flow at every stage between chromatography and contact with the sniffer has resulted in resolution at the sniff port approaching that of the capillary column itself. Furthermore, the selective nature of olfaction itself allows the separate detection of highly overlapped peaks generated by stimulants with different odor qualities (9). Indeed, the receptiveness of the GC/O to a few highly potent odorants makes it one of the most sensitive GC detectors available. However recording and processing datafroma GC/O poses an entirely different set of problems than those faced with other GC detectors
character. However, it is an axiom of analytical chemistry that RT alone is seldom sufficient to identify an unknown chemical, and other properties must be determined in order to associate chemical structure with GC/O data. Finally, as with any other identification procedures, standards must be used to authenticate tentative identifications. Generatedfromthe precepts constructed in the mind of the sniffer encoun-
can be
performed safely (10-12) (Unlike most took used by the analytical chemist GC/O reniiires the exposure of humans
to chemicais, and mis raises safety issues t
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for the chemist. With food extracts, a general rule would be to design the tests so il
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T h e nature of GC/O d a t a
GC/O data involve measurement of two variables: perceived odor activity and chromatographic retention time (RT). The RT is characteristic of chemical properties of the odorant (volatility above a substrate) and has considerable identifying power, especially when combined with other properties of the eluting chemical, such as mass or Fourier transform spectra, RT on a different GC phase, or odor 172 A
Figure 3. A t y p i c a l AEDA c h r o m a t o g r a m s h o w i n g t h e d i l u t i o n v a l u e s a n d o d o r a n t s i d e n t i f i e d in s t r a w b e r r y . (Data from Ret. 26.)
Analytical Chemistry News & Features, March 1, 1997
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matogram where questionable chemicals might elute.) One method of enhancing the usefulness of retention datafromGC/O is to
report standardized times or retention indices (RIs). Championed by Kovats in the 1960s, converting RT to RI values involves the expression of retention in terms of a ratio of the RT of an analyte to the RT of a standard. Retention scaling based on the Kovats method requires the chromatographic separation of a homologous series of normal paraffins, esters, and so on, producing an index that is then the ratio of the RT of an analyte minus the RT of a less-retentive standard to the RT difference between a less-retentive standard and the next most-retentive standard For the past 20 years the most useful resource in laboratories using GC/O has been Jennings and Shibamoto's 1980 rnmpilatinn of RIs for flavor rnmpounds (13) or similar Hntahacpc Hpvplnnprl in the laboratory
The odor activity detected in GC/O is a more complicated variable to assess and record than RT (14). Odor activity is a vector with component odor qualities experienced over a range of intensities at different potencies. This divides the problem of odor measurement into three areas: odor-quality identification, odor-intensity assessment, and odor-potency quantification. The study of the relationship between odor intensity and stimulant concentration called psychophysics has roots in the 19th century and follows an established set of theories experimental protocols and literature
perception it renders at a given concentration, whereas its potency relative to some other stimulant is a comparison of their dose levels at the same intensity response. Both methods for quantifying GC/O data have been used. OSME (17), derived from the Greek word meaning smell, refers to the quantification of perceived intensity, whereas Aroma Extraction Dilution Analysis (AEDA, 18) and CharmAnalysis s19) are based on the measurement of potency. In these methods, the iso-intensity level is the threshold. A discussion of features that distinguish these methods has been discussed in the literature (20,21). Recording GC/O d a t a
The most common form of GC/O data is the association of an odor label with a particular RI region of a chromatogram. Traditionally recorded by simply writing the label for the odor on a chart paper as the sniffer experienced them eluting from the chromatogram, the process has evolved into computerized systems that record the
index range of the odor experience and an associated word chosen from a list presented to the sniffer during the test. When a sniffer is presented with an authentic standard and is provided with experience associating the label with the standard and aid in recalling the label, sniffing ".. .permits ready identification of scores of substances" (22). More recently developed procedures for recording the relative potency of odorants, as well as their odor character, have been used as an aid in deciding which odors are more likely to contribute to precept formation. The assumption is that the odor precept formed when a person smells a mixture is related in some way to the odorants detected separately during GC/O. Unfortunately, the additive effects of compounds with similar odors, aliphatic esters for example, are an obvious challenge to this assumption. Furthermore, the complete volatilization of extracted compounds such as vanillin, which have negligible volatility in most food systems,
The most relevant psychophysical concept to GC/O is the compressibility of the dose-response behavior of olfaction. For example, if you express perceived odor intensity as a value *F and stimulant concentration as O, it has been determined that ¥ follows Steven's Law, ¥=0", where n is usually between 0.3 and 0.8. This compression of the dose-response behavior of odor intensity is not unlike the compressibility of percent transmission in absorption spectroscopy in which a mathematical transformation is required to produce an optical density that is then a linear function of concentration In a similar fashion GC/O data can be somewhat linearized by using transon Steven's Law (15 16); transformations however is limited by the broad range of the exno'nent n exhibited by different odorants stimulating different people
The odor intensity of a stimulant is the
Figure 4. GC/O and GCFID of a Freon 113TM extract of brewed Oolong t e a . (Adapted with permission from Ref. 28.) Analytical Chemistry News & Features, March 1, 1997 173 A
Report results in an exaggerated estimate of their affins as standards. Although similar to AEDA plots, Charm chromatograms are potency, although headspace techniques plots of dilution values versus RI procan be used to minimize this problem. duced when all the on-off responses of Despite these biases, measuring relative the sniffer are added and transformed to potency represents an excellent method account for the dilution factor. Ideally, the for sorting out the small number of conheight of a peak in a Charm chromatotributing odorants from the very large gram is the same as that of a peak in an number of odorless volatiles found in AEDA chromatogram. Charm values are most natural products, fragrances, and the areas of the peaks in the chromatoprocessed foods (23-27). gram and are assumed to represent the Based on the process of sniffing serial ratio of the amount of the compound in dilutions, the two most commonly used the sample divided by an odor threshold methods for measuring odor potency durfor the compound Clearly compounds ing GC/O are AEDA and CharmAnalysis. without odor or those present below an In both methods, samples are diluted and odor threshold concentration will not orothe presence or absence of odor is reduce responses in the chromatogram corded for each dilution (Figure 2). In the Distinguishing the odor-active peaks from case of AEDA, the potency is defined as the mostly odorless ones is an iterative dilution where a particular odor is last deprocess of identification and testing with tected. For example, Figure 3 shows an AEDA chromatogram for fresh strawberry. authentic standards that often remiire tin Figure 4 shows a typical GC/O chromatogram of an extract of Oolong tea proThe odor spectrum duced by CharmAnalysis compared with the FID chromatogram also transformed A serious problem with the display of from a timescale to an RI scale using parGC/O data is caused by the psychophysi,
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Figure 5. Odor spectrum and Charm chromatogram of Oolong t e a data. (Adapted from Ref. 28.)
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Analytical Chemistry News & Features, March 1, 1997
cal compression of the perceived intensity of odors. OSME addresses this problem through the recording of perceived estimates of odor intensity during GC/O. Alternatively, an estimate of the impact of compression on odor potency can be made by transforming the dilution values from an AEDA chromatogram or the charm values from a charm chromatogram using a Steven's Law exponent. Because the reported exponents for odorants from 0.3 to 0.8 a simple compromise would be the square root n = 0 5 Otherwise a different exponent for each peak should be used For example a combination of the OSME and CharmAnalysis methods has been used to obtain estimates of the individual exnonents for nrtorants in racnhprrv (1K\
An additional refinement to the use of Steven's Law for expressing GC/O potency data is shown in Figure 5. Called an "odor spectrum", this transformation normalizes the potency data to 100% in the same way that a mass spectrum is normalized to the most abundant fragment produced in a spectrometer. Odor spectra are more or less independent of the method used to determine potency and the total size of the sample analyzed, andtiieymore closely reflect the contribution of the odorants than the typical GC/O chromatogram does. The odor spectra of headspace samples would be even closer to the pattern of potencies reaching the olfactory epitiielium Future developments and applications In addition to improving the human interface, die most important addition to GC/O technology is an accessible database of odor spectra of commonly encountered natural products, the RIs of component odorants, and a lexicon of descriptors and standards to associate with them. Future applications of GC/O to the study of human perception and diversity have many potential benefits. For example, human subjects in psychophysical or sensory experiments could be quickly assessed for anosmias trained to use lexicons and tested by simply having them sniff the "pure" Gaussian doses in the GC/O effluents of appropriately constructed standards As GC/O technology becomes simpler automatic and less expensive it will not only be useful to study die chemistry that causes human
(25) Guth, H.; Grosch, W. Flavour Fragrance J. Terry E. Acree, Professor of Biochemistry in 1993,8,173-8. the Department ofFood Science and Tech(26) Schieberle, P. In Trends in Flavour Renology at Cornell University, performs research; Maarse, H., van der Heij, D. G., search in isolating and identifying odorEds.; Elsevier: Amsterdam, 1994; Voll 355 active compounds in fruits and vegetables. p. 345-51. (27) Cadwallader, K. R; Tan, Q.; Chen, F.; Address correspondence to Food Research References Meyers, S. M.J. Agric. Food Chem. 1995,Laboratory, Deptt ofFood Science and (1) The Chemical Senses, Moncrieff, R W., Ed.; 43,2432-37. Technology, Cornell University, Geneva, CRC Press: Cleveland, 1967, p. 208-26. (28) King, K. M.. M.S. Thesis, Cornell UniverNY 14456 (
[email protected]). (2) Dravnieks, A In Methods in Olfactory Resity, 1966. search; Moulton, D. G., Turk, A, Johnston, J. W., Eds.. Academic Presss London, 1975, p. 1-62. (3) Fuller, G. H.; Steltenkamp, G. A; Tisserand, G. A Annals. N.Y. Acad. Sci. 1964, 116,711-24. NEW S P E C T R A T R A K ™ 5 7 2 P O R T A B L E G C / M S (4) Acree, T. E.; Nishida, R; Kukami, H. /. Agric. Fooodhem. m.85,33,425-27. (5) Acree, T. E.; Lavin, E. H.; Nishida, R; Watanabe, S. In Flavouu Science And Technology; Bessiere, Y., Thomas, A F., Eds.; John Wiley & Sons: Chichester, 1990, p. 49-52. (6) Cain, W. S.. Cometto-Muniz, ,. C; de Wijk, R A In Science of Olfaction; Serby, M. J., Chobor, K. L, Eds.. SpringerVerlag: New York, 1992, p. 279-308. (7) Dravnieks, A; O'Donnell, A/. Agric. Fooo Chem. 1971,19,1049-56. (8) Acree, T. E.; Butts, R M.; Nelson, R R; Lee, C.Y.Anal. Chem. 1976,48,1821-22. (9) Acree, T. E. In Flavor Measurement, HoH C. T, Manley, C, Eds.; Marcel Dekker, Inc.: New York, 1993, p. 77-94. (10) Mottram, D. S.; Madruga, M. S.; Whitfield, F. B./. Agric. Food Chem. 1995,43, 189-93. (11) Chung, H. Y.; Chen, R; Cadwallader, K. R /. Food Sci. .199, 60,289-91. (12) Boerjesson, T S.; Stoellman, U. M.; Schnuerer, J. L.J. Agric. Food Chem. 1993,41,2104-11. (13) Jennings, W. S.; Shibamoto, T. In Qualitative Analysiisf Flavor and Fragrance Volatiles sy Glass Capillary Gas Chromatography, Academic Press: New York, Compact, lightweight and robust, 1980. he new SpectraTrak 572 gives you the power to perform (14) Abbott, N.; Etievant, P.; Issanchou, S.; Langlois, D.J. Agric. Food Chem. 1993, definitive GC/MS analysis at the point of need. 41, 1698-1703. (15) Roberts, D. D.; Acree, T. E.J. Agric. Food • Ship it to the site, take it on • Hewlett-Packard MSD-based, Chem. 1996,44,3919-25. equipped for rigorous field use. the road, wheel it to the process (16) Fischer, U.; Berger, R G. In Second Pangline... or leave it in the lab! born Sensory Science Symposium. Nobel, • Value-added sampling versatility, A, Ed., 1996, in press. including built-in trap and desorb • Set up and acquire data within (17) Miranda-Lopez, R; Libbey, L M.; Watson, for volatiles, membrane inlet 30 minutes of arrival at site. B. T; McDaniel, M. R/. Food dci. 1992, for real-time direct MS. Analyze 57,985-93,1019. • The timely on-site GC/MS (18) Grosch, W. Trends Food Sci. Technol. soil, water, air, volatiles, solution for expedited site 1993,4,68-73. semivolatiles... (19) Acree, T. E.; Barnard, J.; Cunningham6 characterization, emergency D. G. Food Chem. 1984,14,273-86. The complete GC/MS system ready response, ambient air/air toxics, (20) Etievant, P. X.; Moio, L; Guichard, E.; to go anywhere. Get the full story: forensics, process analytics and Langlois, D.; Leschaeve, I.; Schlich, P.; decision-quality troubleshooting... Chambellant, E. Dev. Food Sci. 1994,35, data, far beyond 179-90. screening or (21) Acree, T. E.; Barnard, J. In Trends in Flasite-seeing. vour Research; Maarse, H., vav ded Heij, D. G., Eds.; Elsevier: Amsterdam, ,994; Vol. 35, pp 211-20. (22) Cain, W. S. Science 1979,203,467-70. INSTRUMENTS CORPORATION (23) Blekas, G.; Guth, H. Dev. Food Sci. 19959 34a, 419-27. fph 1 7(R offt nifY Ch ^ 7
