New Analytical Technologies
Come of Age

October 1995 -- QA/QC

By: Ray Marsili
Contributing Editor

  These days, food laboratories that perform both research and QC analytical testing have an enormous variety of chemical instrumentation from which to choose. New instrument technologies that weren't commercially available just a few years ago are already making invaluable contributions, and new-generation instruments have proven to be extraordinarily simple to use, accurate, reliable and durable.

  The benefits of these new instruments are truly impressive: significantly faster analysis time compared to traditional methods; low cost per analysis; high precision and accuracy; automated control that requires little or no operator attention, freeing technicians to perform other productive tasks; minimal usage of flammable organic solvents and toxic reagents; and advanced, computer-based data management capabilities. With analytical work demand at an all-time high (thanks to the NLEA, HACCP and other regulatory requirements), turning to these modern sophisticated analytical workhorses can obviate the need for hiring new food chemists and lab technicians - an expense that usually far outweighs instrument purchase and maintenance costs.

Combustion protein analyzers

  Named for its inventor, a prominent 19th century European scientist, the Dumas method featured the first apparatus available for the determination of protein in foods. In the Dumas method, a sample is burned in an oxygen-rich atmosphere, the amount of nitrogen gas is measured, and the total protein present is calculated from the nitrogen content. Although the technique initially appeared promising, it proved imprecise and inaccurate and was soon abandoned.

  Soon after the demise of the Dumas test, the Kjeldahl method was developed and it has been used, practically unchanged, for nearly a hundred years as the official test for total protein in foods. The steps involved in Kjeldahl analysis include sample digestion in boiling sulfuric acid, neutralization with sodium hydroxide solution, distillation of the resulting ammonia gas into a trapping solution, titration with an acid solution and, finally, determination of the amount of nitrogen and protein by calculation. The entire process may require over four hours and, excluding the digestion step, require direct technician involvement. Handling flasks of boiling sulfuric acid and the addition of concentrated caustic solution to hot sulfuric acid make it one of the most dangerous tests to perform in the food lab. Furthermore, Kjeldahl tests generate toxic waste since mercury or selenium catalysts are used during the digestion step.

  Despite these limitations, the Kjeldahl test remains the industry standard for protein determination, largely because it has proven to be precise and accurate for nearly all types of food samples. But there is strong indication that Kjedahl's dominance as the favored test method may soon be over.

  The Dumas method, which was abandoned long ago in favor of the Kjeldahl method, is making a comeback in a new and vastly improved form. For example, the operation of St. Joseph, MI-based LECO Corporation's FP-2000 protein analyzer uses the following sequence of operation: Samples (up to 4 grams) are weighed into ceramic boats and loaded into the instrument, where they combust in the pure oxygen environment of the furnace. After passing through a thermoelectric cooler to drop out water vapor, combustion gases are collected in a 4.5-liter volume ballast and a 10-cc aliquot is taken. The gasses are scrubbed and all nitrogen oxides are reduced to nitrogen gas. Each analysis is completed in approximately four minutes.

  With the combustion technique, analysis time is only three to four minutes compared with two to four hours for Kjeldahl; no hazardous waste is generated; cost per analysis ranges from 37 to 50 cents (for Kjeldahl it is approximately $1 per test); and incorporation of an autosampler is possible, allowing analysts to run from 23 to 125 samples unattended. The LECO FP-2000 can store up to 32,000 results in an integrated database, and results can be displayed on a high-resolution touchscreen or printed in a report format.

  The combustion technique for crude protein has received AOAC approval for cereal grains and oilseeds, meat/meat products, and animal feed. It is currently being reviewed for dairy products.

Fourier transform IR

  Fourier transform infrared analyzers aren't new. What is new are FTIR instruments designed and optimized specifically for food applications. One example is Foss Food Technology Corp.'s (Eden Prairie, MN) MilkoScan FT 120 dairy product analyzer.

  In the past, dairy processors who wanted to monitor fat, solids, protein and lactose in incoming raw milk and finished dairy products usually had to wait several hours for results. Traditionally, dairy technicians used the Roese-Gottlieb (Mojonnier) test for butterfat (a solvent extraction/gravimetric method), the Kjeldahl method for protein determination, and HPLC analysis for lactose. Using the MilkoScan FT 120, all these results can be obtained with the same instrument in less than one minute. The FT 120 can analyze raw and processed milk, processed cream, butter, yogurt, cheese, whey, ice cream, sweetened condensed milk, and many other types of dairy products.

  Using this rapid technique, a small amount of dairy product is placed in the instrument which contains an infrared radiation source. The spectrometer scans the amount of radiation that passes through the sample over a given frequency range and records the percentage of radiation that is transmitted. Radiation absorbed appears as a band in the spectrum.

  Particular types of chemical bonds usually stretch and bend within certain narrow frequency ranges. For example, protein molecules are basically amino acids joined in a long chain by peptide bonds. The absorption exhibited by proteins at 6.5 mm is due to bending vibrations in the nitrogen-hydrogen bonds (also called peptide bonds). Thus, the measurement represents the number of amino acids rather than their weight. However, since the composition of protein in milk is fairly constant, this causes no significant problem. In contrast to the standard Kjeldahl method, the 6.5 mm measurement does not include non-protein nitrogen.

  By using an FTIR interferometer to scan the full infrared spectrum, the FT 120 also can measure the intensity of absorbed radiation corresponding to specific bonds for carbohydrates, fats and other chemical components of the food material. Preliminary studies indicate the instrument can quantitate specific sugars in sugar mixtures (e.g., dextrose, fructose and lactose) and perhaps even casein in the presence of other dairy proteins (whey proteins).

  Unlike near infrared (NIR) instruments, FTIR instruments don't require constant calibration with standards. In fact, the instrument comes with reliable factory-installed calibrations for milk, cream and other dairy products. The PC Windows-based software is user-friendly, but powerful and flexible enough to be customized for non-standard applications. In the future, FTIR technology is likely to be applied to nondairy food testing, as well.

Capillary electrophoresis

  Capillary electrophoresis (CE) is a family of analytical methods that employ narrow-bore (10-200 mm internal diameter) capillaries to perform high-efficiency separations of both large and small molecules. In some respects, it can be roughly compared to a combination of HPLC and electrophoresis. High voltages are used to separate molecules based on differences in charge and size. Various CE techniques can be used to perform separations based on several different physical and chemical mechanisms, including molecular size (sieving), isoelectric focusing, and hydrophobicity.

  Commonly, detection is achieved by monitoring UV absorbance directly on-line through a window in the capillary. Recent detector options include diode array, mass spectrometry and laser-induced fluorescence (LIF). Beckman Instruments Inc.'s second generation CE system offers LIF detector capability with an unprecedented 500 fold increase in sensitivity over UV detection.

  CE technology has been used to quantitate specific proteins, vitamins, organic acids, amino acids, flavonoids, nucleotides, anthocyanins (in cranberries), and carbohydrates in a variety of food matrices. Compared to other chromatographic techniques, CE usually requires less sample, less sample preparation, and shorter analysis time. Unlike Kjeldahl, combustion and FTIR, CE techniques can quantitate individual protein types in complex mixtures of proteins.

  One application of CE is the determination of goat milk in cow milk. When the price of goat milk drops below that of cow milk, the risk of fraudulent addition of goat milk to cow milk exists. Bovine milk proteins consist of approximately 80% caseins (as (s1-, (2-, (-, and (-casein), which exist in milk as a micellar complex in the approximate proportions of 4:1:4:1. The remaining 20% of the milk proteins are whey proteins, predominantly (-lactoglobulin and (-lactalbumin. The protein profile of goat milk is significantly different. Inspection of CE electropherograms of cow milk can readily show whether the sample has been illegally diluted with small amounts of goat milk.

  Traditionally, milk proteins have been separated by gel electrophoresis, a technique that is tedious, time-consuming and not amenable to quantitation (even with densitometers). Recently, ion exchange and reverse-phase HPLC have been employed. However, these techniques also involve lengthy analysis times and don't offer the resolution of CE.

  Dr. Rafael Jimenez-Flores at the Dairy Science Department of the California Polytechnic State University (San Luis Obispo, CA) uses CE to monitor milk proteins and milk protein quality. According to him, the technique is particularly amenable to the analysis of peptides, with analysis times of only 10 to 20 minutes. By examining CE peptide profiles, he often can determine if poor quality milk samples have undergone proteolysis due to microbial contamination, plasmin (a milk enzyme that hydrolyzes peptide bonds), or improper milk handling.

  "CE can be used as a rapid technique for monitoring lactic acid and other organic acids produced during the processing of cultured dairy products," says Jimenez-Flores. The ultimate goal of this type of testing is to improve control of fermentation during processing. Jimenez-Flores is also investigating various types of CE columns to improve the resolution of dairy proteins (e.g., to be able to differentiate genetic variants).

  Dr. Eileen LeBlanc, assistant professor at the Department of Agricultural Food and Nutrition Science, University of Alberta (Edmonton), Canada, is also an advocate of CE for food protein analysis. She uses CE with UV and LIF detection for analyzing proteins and nucleotides in fish. With the technique, LeBlanc monitors protein composition to distinguish fish species and checks peptide profiles and nucleotide ratios to determine fish quality. Both of these applications are becoming increasingly important as more and more fish are being imported by Canada and the United States.

  Identification of specific enzymes that cause fish protein degradation is possible with CE, according to LeBlanc. She has used CE to monitor the effectiveness of various modified-atmosphere packaging (MAP) parameters to preserve fish. She uses zonal CE to monitor sarcoplasmic proteins (water soluble proteins) and SDS-gel CE methods for quantitating myofibrillar proteins (salt soluble proteins).

  LeBlanc predicts that more analyses will be done by CE as instrument costs continue to drop. "Gel electrophoresis requires 48 hours, while protein analysis by CE can be completed in 30 minutes," says LeBlanc. "The technique is sensitive, rapid and requires only nanoliters of sample. Unlike HPLC which uses hundreds of milliliters of organic mobile phase, CE requires only a few milliliters of buffer solution."

Supercritical fluid extraction

  Supercritical fluid extraction (SFE) is a sample preparation technique that uses supercritical fluids (usually carbon dioxide) instead of normal organic solvents like diethyl ether and methylene chloride as extracting solvents. Carbon dioxide gas is transformed into a supercritical fluid when both the temperature and pressure are equal to or exceed those of its "critical point" (31°C and 73 atm for carbon dioxide).

  Supercritical fluids have densities and solvating powers similar to liquid organic solvents but have rapid diffusion characteristics and viscosities similar to those of a gas. The combination of liquid-like solvating power and gas-like diffusivity make supercritical fluids ideal reagents for extracting organic analytes from complex food materials. Extraction characteristics (i.e., solvent strength) of supercritical fluids can be varied and controlled by adjusting pressure, temperature or both, and by employing "modifiers." For example, if pressure and temperature increases alone don't dissolve the analyte of interest, a small amount of modifier such as methanol, isopropanol, acetonitrile or water can be added to the food material either before extraction (static addition) or continuously during extraction (dynamic addition) to increase the solvating power and the polarity of the supercritical fluid.

  SFE is particularly well suited for high-solids foods like snack foods. In the case of semisolid foods (e.g., ice cream and pudding), the excess free moisture is bound up with an inert solid support (e.g., diatomaceous earth or hydromatrix) before the sample is added to the extraction thimble. Analysis of fluid samples like milk is problematic. However, new sample manipulation techniques will undoubtedly solve these problems in the near future.

  Commercial SFE instruments have been available for the past few years, but only recently have instrument improvements in decompression control and variable restrictor design made the technique reliable and practical enough for routine QC food testing. In the past, the major limiting factor in automating SFE for QC applications was the irreproducible flow caused by clogging of fixed restrictors. By electronically sensing plug formation and automatically opening or closing the restrictor to maintain uniform flow rates, extraction conditions can now be precisely controlled and reproduced in today's state-of-the-art SFE instruments.

  Compared with traditional solvent extraction techniques, SFE greatly reduces the use of toxic and flammable organic solvents, enables automation, generates little waste, and significantly reduces analyst time, space and glassware requirements. Furthermore, by manipulating instrument conditions such as carbon dioxide density, temperature, modifier, analyte trapping techniques, and so on, the analyst can perform selective extraction of specific chemical analytes from a complex matrix - e.g., cholesterol from butterfat.

  SFE has been used for extracting (-carotene from vegetables, cholesterol from egg powder, pesticides from fruits and vegetables, flavor compounds from spices, and fats and oils from meats, nuts and other food products. After SFE, the analyte concentration is usually determined by an appropriate chromatographic technique. Probably the most used SFE food application to date is the determination of total fat in animal feed and snack foods.

  One of the world's largest snack food companies recently purchased a large number of SFE instruments to determine total fat content of snacks, according to Athos Rosselli, product manager for Suprex Corp., Pittsburgh. By using SFE and gravimetric analysis, the company can complete the analysis in less than one hour - significantly less time than the eight hours previously required for Soxhlet extractions. The company uses SFE analysis to calibrate its on-line near infrared analysis unit. The company requires rapid, accurate fat analysis for better control of processing and for meeting label requirements. Suprex's FatMaster SFE system offers high throughput via dual-vessel simultaneous extraction, providing four results per hour for production control needs. The system requires minimal training to operate and can be used for a variety of other applications.

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