Advances in Flavor Sample Preparation

Flavor is a major factor in the consumer’s perception of the quality of a food product. If it doesn’t taste good, no one will buy it. Measuring flavors, however, is a complex task because it involves measuring not only the flavor itself, but also measuring how a consumer reacts to a flavor. So flavor measurement is a combination of sensory evaluation with chemical and physical instrumental analysis. The active components of a flavor compound are detected by a combination of gas chromatography and olfactometry (GC/O). However, instrumental analysis is a last step in the analytical procedure that starts with sample preparation. This article will discuss some of the latest advances in techniques for this critical first step in the analysis of food flavors.

Techniques
There is no universal isolation method for the entire spectrum of volatile flavor compounds. But a variety of established isolation techniques are available. These include liquid/liquid extraction, liquid/solid extraction, solid-phase extraction, accelerated solvent extraction, headspace analysis, gas extraction, direct thermal desorption, and others. These techniques have been used for many years, but other sample preparation methods have been used recently:
• Solid-Phase Microextraction. First reported on and developed in the late 1980s and early ‘90s, solid-phase microextraction (SPME) is increasingly being used for the gas chromatographic determination of a wide variety of volatile and semivolatile organic compounds in water or aqueous extracts of different substrates.

It involves the extraction of specific organic analytes directly from aqueous samples, or from the headspace of these samples in closed vials, onto a fused-silica fiber coated with a polymeric liquid phase, poly(dimethylsiloxane) or polyacrylate. After equilibration, the fiber containing the absorbed or adsorbed analyte is removed and thermally desorbed in the hot injector of a gas chromatograph. The analytes are then analyzed by GC using an appropriate column and detector, with or without cryofocusing.

The technique is simple and fast, and does not employ any organic solvents for either sample preparation or cleanup. This is desirable because, unlike other methods, it does not release polluting organic solvents into the environment. The technique has been successfully applied to the determination of a variety of compounds in flour, tea, coffee, spices, beer, wine, fruit juices, fruit drinks, and milk. SPME may also be combined with other chromatographic and spectroscopic techniques such as thermal desorption or mass spectrometry.

• High-Pressure Extraction with Supercritical CO₂. Using supercritical carbon dioxide has been an established industrial-scale technique for several years. When pressurized CO₂ is heated above a certain critical temperature, it becomes a supercritical fluid, which has some of the properties of a gas and some of a liquid. The fact that it behaves like a gas means that it can easily penetrate into a sample and extract the lipids, while the fact that it behaves like a fluid means that it can dissolve a large quantity of lipids (especially at higher pressures).

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Instruments based on this principle heat the food sample to be analyzed in a pressurized chamber and then mix supercritical CO₂ fluid with it. The CO₂ extracts the lipid and forms a separate solvent layer, which is separated from the aqueous components. The pressure and temperature of the solvent are then reduced, which causes the CO₂ to turn into a gas, leaving the lipid fraction remaining.

An industrial-scale example of this technique is the decaffeination of coffee and tea. More recently, this extraction method has been applied to the isolation of flavor and fragrance chemicals from natural products. Because of its mild extraction conditions, the method is especially suited to isolating essential oils from spices, flowers, herbs, leaves, seeds, and roots. Like SPME, the method has the advantage of eliminating the use of organic solvents.

• Solvent-Assisted Flavor Evaporation. Wolfgang Engel, Wolfgang Bahr, and P. Schieberle in 1999 developed a new distillation technique, called solvent-assisted flavor evaporation (SAFE), for the extraction of flavor volatiles from complex aqueous matrices, such as beer, fruit juices, milk, and cheese. In this technique, the distillation vessel and transfer tubes are kept at low temperatures (20–30°C) to avoid condensation of compounds with high boiling points, and the sample is added by dropping aliquots from the funnel into the vessel to reduce time of extraction.

The developers pointed out that the technique provides higher yields of volatiles, polar flavor substances, and odorants from fat-containing substances; allows the direct distillation of aqueous samples such as milk, beer and orange juice; and recovers flavor samples with organoleptic properties as close as possible to those of the native product.

Instruments and Accessories
The following are some instruments and accessories available for flavor measurement:
• Evaporative Light Scattering Detector, the ELSD 800, may be used to replace or complement existing detectors on liquid chromatography systems. The unit may be used for detecting and quantifying non-volatile and semi-volatile analytes in a sample. Historically, these detectors have been used for applications such as lipids, carbohydrates, fatty acids, and amino acids. According to the manufacturer, the detector gives a closer representation of sample mass than UV detectors and can be used in parallel with mass spectrometry (MS) to obtain maximum structural and concentration information. For more information, contact Alltech Associates Inc., 2051 Waukegan Rd., Deerfield, IL 60015 (phone 800-255-8324; fax 847-948-1078; www.alltechWEB.com).

• GC-Olfactometry System, the Sniffer 9000, is designed to be a dedicated, stand-alone “sniffing-device” to be connected to any gas chromatograph. In the past decades, many detection techniques have been hyphenated to gas chromatography. Less attention has been paid to GC/O, in which the human nose plays the role of the detector. However, the human nose is often more sensitive than any physical detector, and GC/O exhibits powerful capabilities that can be applied to flavors and perfumes, as well as to any odoriferous products (e.g., pollutants).

Olfactometric (or “sniffing”) techniques allow the determination of impact odorants in food. They can be classified into two categories: dilution methods, which are based on successive dilution of an aroma extract until no odor is perceived at the sniffing port of the chromatograph; and intensity methods, in which the aroma extract is only injected once but the panelist records the odor intensity as a function of time. For more information, contact Brechbuehler Inc., 18512 Carrot St., Ste. 409, Spring, TX 77379 (phone 281-370-9290 fax 281-370-9259; www.brechbuehler.com).

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• Purge-and-Trap and Headspace Instruments may be used for monitoring flavor volatiles in food packaging and other food and beverage applications. The new Purge and Trap Velocity XPT™, is said to be ideal for flavor profiling of volatiles in food and beverages. The unit is a patent-pending accelerated purge-and-trap system with a forward-focusing chamber that provides efficient desorption, resulting in high recoveries and lowest carryover, especially for higher-boiling compounds. The sample pathway is said to prevent loss of active, polar, or high-boiling compounds. Other time-saving features include electronic flow control, pressure monitoring, and automatic leak check. For more information, contact Teledyne Tekmar, 4736 Socialville-Foster Rd., Mason, OH 45040 (phone 513-229-7000; fax 513-229-7050; www.tekmar.com).

• Electronic Nose, the zNose, is said to be the only electronic nose with the ability to speciate and measure the concentration of individual chemicals in 10 sec. Based on flash chromatography, the unit creates chromatograms, virtual chemical sensors, and/or 2D images. The portable design allows for use in the field or the laboratory. The unit may be used as a screening tool to reduce the number of negative laboratory results. For more information, contact Electronic Sensor Technology, 1077 Business Center Cl., Newbury Park, CA 91320 (phone 805-480-1994; fax 805-480-1984; www.estcal.com).

• Gas Chromatograph, the GC-17A, is designed to provide the parameter control necessary for the most applications and schedules. The system ensures that all the operating parameters are set reproducibly, including carrier gas flow, split ratio, all heated zone temperatures, detector combustion and make-up gas flows, and detector output range and current. Analyses are achieved with electronic flow control of all carrier gas and detector gas functions. Flow control of the carrier gas is combined with pressure control of the detector gases (up to 14 flow channels). Up to ten GC parameter files can be entered, and the GC can be directly controlled with the intuitive keypad. Parameter data are carefully protected by a password system which limits access to critical settings. The user may set parameters with a GC clock scheduler. For more information, contact Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046 (410-381-1227; fax 410-381-1222; www.ssi.shimadzu.com).

• SPME is offered as an accessory to the Alpha MOS Sensor Array, MS, and electronic nose instruments. The accessory is said to offer better sensitivity; lower detection limits; enhanced selectivity, with the possibility of choosing between classes of fibers; stronger reproducibility, and faster analysis (2–15 min on average). For more information, contact, Alpha MOS America, 33 N. River St., Hillsborough, NJ 08844 (phone 908-359-9396; fax 908-359-9398).

• Electronic Nose, the Cyranose® 320, has applications in the food, packaging, and flavor industries. It may be customized for the specific application. The unit is trained by measuring vapors representative of the process. These measurements create a database of digitized patterns. All future measurements are compared to these patterns to identify the vapor. The unit’s polymer composite sensors have been shown to respond to a wide range of organic compounds, bacteria, and natural products. For more information, contact Cyrano Sciences, 73 N. Vinedo Ave., Pasadena, CA 91107 (phone 877-744-1700; fax 626-744-1777).

by JAMES GIESE
Internet Editor